Conditioned medium from MC3T3-E1 was used for osteoblast stimulation of CRPC cells. The capacity of LNCaP-19 cells to induce sclerotic lesions was assessed in intratibial xenografts and verified by serum markers, histological analysis and bone mineral density (BMD) measurements. The CRPC cell line LNCaP-19 expresses a pronounced osteogenic profile compared to its parental androgen-dependent cell line LNCaP. Osteoblast-derived factors further increase the expression of genes known to enhance metastatic progression of PC. LNCaP-19 forms sclerotic tumors in tibia of castrated mice as evident by increased total BMD (P < 0.01). There was a strong correlation between serum osteocalcin and BMD . For the first time we demonstrate that a CRPC cell line generated in vitro has osteogenic capacity and that osteomimicry can be an inherent feature of these cells. Osteoblast-derived factors further promote the osteogenic and metastatic phenotype in CRPC cells. Altogether, our model demonstrates that both tumor cells and osteoblasts are mediators of the bone forming process of CRPC.Castration-resistant prostate cancer (CRPC) is strongly associated with sclerotic bone metastases and poor prognosis. Models that mimic human CRPC are needed to identify the mechanisms for prostate cancer (PC) growth in bone and to develop new therapeutic strategies. We characterize a new model, LNCaP-19, and investigate the interaction between tumor cells and osteoblasts in the sclerotic tumor response of CRPC. Osteogenic profiling of PC cell lines (LNCaP-19, LNCaP, C4-2B Today there are limited therapeutic alternatives for patients with advanced prostate cancer (PC), and androgen deprivation therapy (ADT) is the primary option. ADT is initially highly effective in suppressing tumor growth. However, it leads to increased bone degradation resulting in the release of growth factors from the bone matrix, many of which can stimulate prostate cancer (PC) cell attraction and tumor growth in bone –3. The oBone metastases behave differently depending on their tumor origin. Metastases from breast and lung cancers are primarily osteolytic, inducing bone degradation , 4, 5, wSkeletal metastasis is the consequence of the bidirectional interplay between tumor cells and bone cells. Metastatic PC cells produce a variety of bone-stimulating factors including prostate specific antigen (PSA), endothelin-1, bone morphogenetic proteins (BMPs) and insulin-like growth factor 1 (IGF-1), that have direct or indirect effects on bone formation –11. MoreTumor cells that preferentially metastasize to bone acquire features normally expressed by bone cells, particular to the osteoblastic phenotype, an adaptation referred to as osteomimicry –17. The The understanding of the bone metastatic disease, and hence, the development of efficient and specific treatments is limited due to the difficulty of obtaining samples from bone metastases from patients. Therefore, models that mimic the human disease are critical for elucidation of alterations in both tumors and bone cells and for evaluation of new therapeutics. Today, few models exist to study the osteoblastic function of CRPC, for example LuCAP-23.1 and MDA-PCa 2a and 2b form osteoblastic lesions while C4-2B, IGR-CaP1 and PCSD1 give mixed osteolytic/osteoblastic lesions –22.The overall aim with this study was to characterize a novel model, LNCaP-19, for studies of the osteoblastic process in metastases of CRPC under castrated conditions reflecting the clinical situation. LNCaP-19 was established in our laboratory as an in vitro derived castration-resistant subclone of LNCaP . It has We here show that LNCaP-19 is suitable for studies of the osteoblastic function of CRPC, in a bidirectional system both in vitro and in vivo. We demonstrate that osteomimicry can be an inherent feature of PC cells, which is further potentiated by the acquisition of castration-resistance. We also show that osteoblasts trigger a more pronounced osteogenic and aggressive phenotype of the CRPC cells and initiate a cross-talk between tumor- and bone cells. Importantly, this study demonstrates that both tumor cells and osteoblasts are potential mediators of bone formation.P < 0.05) compared to control cells treated with L19CM and FCM. This OCM-specific stimulation was only seen in LNCaP-19 since both OCM and FCM stimulated LNCaP proliferation 1.8 times compared to LNCaP-CM (data not shown).It is known that bone metastases are a result of the communication between bone and cancer cells and that PC cells acquire bone-like properties during disease progression. Therefore, in the present study, we investigated the ability of the LNCaP-19 cells to mineralize extracellular bone matrix. After 21 days of culture in steroid-depleted medium, both promineralization medium (PM) and osteoblast-conditioned medium (OCM) induced mineralization in LNCaP-19 compared to control medium (αMEM) or fibroblast-conditioned medium (FCM)  and the osteolytic castration-resistant cell line PC-3. All four cell lines expressed detectable levels of the majority of the genes included in the osteogenesis array, such as the transcription factor of the osteoblastic lineage msh homeobox 2 (MSX2) as well as bone morphogenetic protein (BMP)-1, -4 and -6, SMADs and osteonectin  (Table RUNX2) and alkaline phosphatase (ALPL) were expressed in LNCaP-19, LNCaP and PC-3 but absent in C4-2B4.As the castration-resistant LNCaP-19 was able to mineralize, an osteogenesis gene signature array was used to identify the underlying gene program responsible for its osteomimetic properties. The osteogenic phenotype was compared between four different PC cell lines, LNCaP-19, mixed osteoblastic/osteolytic LNCaP (androgen-dependent) and C4-2B4 are both derived from LNCaP and display osteogenic phenotypes. Comparing the osteogenic potential between these cell lines, LNCaP-19 exclusively has a basal expression of osteoblast-cadherin (CDH11), collagen 1a1 (COL1A1) and osteopontin , all vital genes for tumor interaction with osteoblasts and osteoblastic activity. Despite the different status in androgen dependence, LNCaP and C4-2B4 share a similar gene expression pattern, such as the basal expression of osteocalcin , fibroblast growth factor receptor (FGFR), insulin-like growth factor 1 (IGF1) and transforming growth factor, beta 2 (TGFB2). However, LNCaP-19 and C4-2B4 both have a basal expression of dentin sialophoshoprotein (DSPP), a gene associated with aggressive PC and mineralization process of enamel, which was absent in LNCaP.LNCaP-19 and C4-2BCSF2), twist homolog 2 (TWIST2) and BMP3, were exclusively expressed in PC-3.A distinct pattern of genes involved in osteoclastogenesis and inhibition of bone formation, including colony stimulating factor 2  both associated with osteoblast communication and bone-tropic metastasis of PC. These genes were undetected in LNCaP. Furthermore, two genes were downregulated in LNCaP-19, collagen 16a1 (COL16A1) and matrix metalloproteinase-13 . Importantly, these genes are associated with chondrocytogenesis and osteoclastogenesis, respectively. The expression of the bone matrix proteins Osn and Opn was shown to be upregulated OCM-stimulated LNCaP cells, while their expression levels were unchanged in LNCaP-19. C4-2B4 was less responsive to OCM stimulation. However, C4-2B4, which lacks detectable basal expression of RUNX2, responded to OCM with induction of this transcription factor. Unique for C4-2B4 was the induction of fibroblast growth factor 1 and -2 after OCM stimulation.The osteogenesis gene signature array was further used to detect differently expressed genes in LNCaP-19, LNCaP, C4-2BBMP2, an inhibitor of bone formation. In addition, significant downregulation of the Bmp2 antagonist MGP (matrix Gla protein) levels were demonstrated in OCM-stimulated PC-3 cells.None of the osteoblast-associated genes were changed in the osteolytic PC-3 cells in response to OCM stimulation. In contrast, PC-3 cells displayed an increased expression level of 4 significantly (P < 0.01) induced proliferation of pre-osteoblasts to the same extent as PM treated cells (P < 0.01), while CM derived from PC-3 and LNCaP had no significant effect on pre-osteoblast proliferation comparable to αMEM control medium  were stimulated with conditioned medium (CM) derived from CRPC cell lines. The osteoblastic differentiation process is well characterized and consists of distinct phases of proliferation, matrix maturation and mineralization. CM with soluble factors derived from LNCaP-19 and C4-2BAlpl, which was sustained over 21 days, a pattern similar to C4-2B4. LNCaP-19 also induced Ocn expression at day 3 (~2.9 fold) compared to αMEM treated control cells, but the mRNA expression declined to control levels at day 7. Expression of endogenous osteoprotegerin (Opg), an inhibitor of osteoclast activation, was elevated by LNCaP-19 stimulation at day 3 and sustained upregulated at day 7. The main transcription factors of the osteoblastic lineage, Runx2, Osx and Msx2 were not affected in MC3T3-E1 by CM stimulation (data not shown). In a control experiment, NIH3T3-E1 fibroblasts did not induce any of the osteoblast differentiation markers in response to LNCaP-19 CM (data not shown).To further assess the effect of CRPC-secreted factors on osteoblastic differentiation, we studied the induction of mRNA expression of established differentiation markers in MC3T3-E1 pre-osteoblasts cultured with CM Fig. b. Factor4, while the osteolytic PC-3 and LNCaP did not induce mineralization in osteoblasts. The observed Alp mRNA expression levels  and non-castrated mice (P < 0.028). The sclerotic effect was also demonstrated with a pronounced difference of trabecular BMD to the control tibia, in the castrated group (P < 0.002) and the non-castrated (P < 0.003)  as well as trabecular BMD   (data not shown). The sclerotic response in LNCaP-19 tumors may partly depend on inhibited osteoclast activation and osteolysis, since Opg serum level was significantly elevated in tumor bearing castrated and non-castrated mice compared to control mice (P < 0.002).Increased bone formation was demonstrated by an increase in total bone mineral density (BMD) of the tumor-bearing tibiae compared to the non-tumor-bearing control tibiae in both castrated , which could explain its osteoblastic potential also in steroid deprived conditions. The absence of RUNX2 expression in C4-2B4 in basal steroid deprived conditions is a striking difference, which could possibly be due to the fact that C4-2B4 was derived in vivo, from a bone metastasis from C4-2 [4 in vivo may be dependent on the presence of bone cells to induce RUNX2, which was also demonstrated in vitro in the present study. In contrast, LNCaP-19, which is developed in vitro without influence from bone, has the full osteogenic gene programme activated independent of osteoblast stimulation.To further characterize LNCaP-19 as an osteogenic CRPC model, we compared the bone-associated gene profile of LNCaP-19 with other CRPC cell lines. Compared to both the parental androgen-dependent LNCaP and the in vivo derived osteoblastic castration-resistant cell line C4-2Brom C4-2 . Thus, tBMP3, TWIST2 and CSF2 and -3. This observation suggests that the expression of these genes may prevent the ability of PC-3 to mineralize or to induce mineralization in osteoblasts. Moreover, the bone-forming genes that were observed to be elevated in response to osteoblast-derived factors in LNCaP-19 in comparison to PC-3 cells, suggesting that soluble osteoblast-derived factors increase the osteogenic potential of osteogenic CRPC cells, while the effect on osteolytic cells is minor.The strongly osteolytic cell line PC-3 displayed most of the osteogenic profile but in contrast to LNCaP-19 also expressed a different set of genes strongly associated with osteoblast inhibitory and osteolytic events, such as 4, MDA-PCa 2a and 2b) [The observation that LNCaP-19 has the capacity to mineralize matrix without osteoblast stimulation suggests that osteomimicry can be an inherent property in advanced PC, independent of bone stimulation. Interestingly, LNCaP-19 was derived from a lymph node metastasis and further progressed by androgen deprivation in vitro, and thus has never experienced contact with the bone environment. However, osteogenic capacity has previously been demonstrated in other CRPC cell lines (C4-2B and 2b) , but in RUNX2, the main transcription factor of osteoblast differentiation, which has previously been found to be ectopically expressed in metastatic cancer cells [MMP2 [4 was induced by Runx2 [RUNX2 have an important role in the development of the osteogenic phenotype of CRPC. The relatively weak response of C4-2B4 to OCM stimulation may be a result of the conditions under which it was developed. The growth in bone may have selected for cells that were well adapted to that microenvironment, and as a result an additional OCM stimulation in vitro does not dramatically change its phenotype. However, in vitro OCM stimulation induced expression of fibroblast factor 1(FGF1) and -2(FGF2) exclusively in C4-2B4 cells. Since FGF1 has been shown to prevent osteoporosis in a rat model [Osteoblast-secreted factors induced a phenotypic shift of LNCaP-19 cells, including morphological changes, increased proliferation and capacity to mineralize matrix. Interestingly, this phenotypic shift of LNCaP-19 is associated with elevated expression of genes related to tumor aggressiveness and metastasis of CRPC. Among these was er cells –33. In Pls [MMP2 , 35. Thiby Runx2 . This suat model , this coCDH11 and MMP2 in LNCaP-19 in response to OCM indicates induction of an increased potential for interaction between osteoblasts and PC cells in the formation of bone metastases [MMP2 was identified as a key factor of the vicious cycle of osteoblasts and metastatic tumor cells [CDH11, also known as osteoblast-cadherin, is reported to be highly expressed in bone metastases and bone-derived PC cell lines. Moreover, CDH11 was previously reported to mediate cell-invasion and adhesion between PC cells and osteoblasts [CDH11 are suggested to be partly responsible for the bone-tropism of PC. The expression of MMP2 and CDH11 in LNCaP-19 and PC-3 in comparison to LNCaP and elevated expression levels of these genes upon OCM stimulation in LNCaP-19, suggests that castration-resistance potentiates the communication between tumor cells and osteoblasts.The vicious cycle involving tumor- and bone cells in osteolytic metastases from breast and lung cancer has been well characterized . Howevertastases , 39. Recor cells . The CDHeoblasts , 39. AltOcn on day 3 and maintained expression of Alpl over the differentiation process. Taken together, these findings are in agreement with clinical observations of CRPC metastases, which are composed of a high proportion of immature new woven bone and non-mineralized matrix [In addition to osteoblast-induced changes in CRPC cells, we demonstrate that LNCaP-19 stimulates pre-osteoblasts to differentiate, proliferate and mineralize in a manner similar to other osteoblastic CRPC models . Previoud matrix , which id matrix . Ocn is d matrix . In lineOpg and serum level of Opg in osteoblasts and tumor bearing mice respectively, in response to LNCaP-19, suggest a local suppression of osteoclast activation which could contribute to the overall osteoblastic response in CRPC tumors.Osteoblasts can regulate bone metabolism by modulating the bone resorption process, directly via the production of receptor activator of nuclear factor κβ ligand (RANKL) and negatively via the secretion of its decoy receptor Opg , 46. In Furthermore, the present study demonstrates the capacity of LNCaP-19 to form sclerotic lesions in bone upon intratibial implantation in both castrated and non-castrated mice. Interestingly, LNCaP-19 was shown to induce an osteoblastic response in tibiae of castrated mice with increased BMD. This in contrasts to the commonly used LNCaP bone-derived C4-2 model, which under castrated conditions results in mixed osteoblastic/osteolytic lesions with an overall decrease in BMD . We alsoWith similarity to clinical observations , the LNCIn conclusion, our work presents a model, closely mimicking the clinical situation, for studies of the osteoblastic function of CRPC both in vitro and in vivo. We here show that osteomimicry can be an inherent feature of CRPC cells, which is further promoted by osteoblast stimulation. Moreover, osteoblast-secreted factors trigger the aggressiveness of CRPC cells and potentiate cross-talk between tumor- and bone cells in the sclerotic situation. Collectively these results indicate that targeting the communication between osteoblasts and PC cells could inhibit the progression of bone metastases in CRPC.4 was kindly provided by Professor Thalmann, Dept of Urology, University of Bern, Switzerland. PC cells were routinely maintained in RPMI-1640 supplemented with glucose, sodium pyruvate (PAA), 10 % foetal bovine serum   or 10 % dextrane charcoal-treated FBS  (LNCaP-19). Pre-osteoblasts, MC3T3-E1 cells, clone 4, were obtained from the ATCC . Osteoblasts were maintained in ascorbic acid (AA) free αMEM (Invitrogen) and 10 % FBS. Murine fibroblasts  were maintained in DMEM (Invitrogen) supplemented with 10 % FBS. All cultures were supplemented with penicillin/streptomycin. Passage numbers between 17 and 21 were used for LNCaP-19 cells and passage numbers 5–9 for PC-3 and C4-2B4. For osteoblasts and fibroblasts, passage numbers between 3 and 8 were used in the experiments. Passage number 1 is defined as the first passage in our laboratory.The androgen-dependent PC cell line LNCaP, FGC clone, was obtained from the American Type Culture Collection . LNCaP-19 a CRPC cell line, derived from LNCaP by androgen deprivation, was established in vitro in our laboratory . PC-3 waTo induce mineral production, cells were cultured in promineralization medium (PM) consisting of αMEM supplemented with 10 mM β-glycerophosphate  and 50 mg/ml L-AA (Sigma) or cell-conditioned medium (CM) for 21 days.Cell-conditioned medium (CM) was obtained to assess the effects of secreted factors produced by osteoblasts and CRPC cells on gene expression level, proliferation and mineralization. Cells were seeded in T75 flasks and grown to 70–80 % confluence in their respective media, cells were washed with PBS and medium was replaced with serum-free αMEM. CM was collected after 48 h culture. The cell number was determined and CM was adjusted by dilution in serum-free medium to normalize for differences in cell density. CM was centrifuged to remove cell debris and stored at −20 °C until use. Maximal final concentration of CM used in the experiments was 75 %.4, PC-3, and LNCaP), OCM (osteoblast-conditioned medium from MC3T3-E1) and FCM (fibroblast-conditioned medium from NIH3T3).Abbreviations used: CM  as described above. After 48 h, cells were washed with PBS and standard medium was replaced with αMEM, PM or CM. Cell culture medium for osteoblasts and fibroblasts was supplemented with 10 % FBS. For studies of osteoblastic differentiation markers, cultures were maintained in CM for 3, 7 or 21 days, when cells were harvested for RNA extraction. Medium was changed every 3 days.Osteoblasts were seeded in T25 flasks . Since osteoblasts are dependent on serum for survival in culture, all medium was supplemented with 10 % FBS. LNCaP-19 cells were seeded at a density of 5.0 × 103 cells per well in αMEM with 1 % DCC. Medium was replaced on day two with OCM, FCM, or control medium , supplemented with 1 % FBS-DCC. Proliferation was monitored by the BrdU ELISA colorimetric assay  in accordance with the manufacturer’s instructions after 48, 96 h (LNCaP-19) and 5 days (MC3T3-E1). Stop solution (1 M H2SO4) was added and plates were read in a Wallac 1420 Victor3™ multiplate reader  at a 450 nm with reference filter at a 690 nm.Osteoblasts were seeded in 96 well plates at a density of 3.5 × 105 or 2 × 105 cells per well in αMEM with 10 % FBS or RPMI-1640 with 10 % FBS-DCC, respectively. After 48 h, medium was changed to CM, OCM or PM and mineral deposition was determined by von Kossa or alkaline phosphatase staining after 21 days. Briefly, for von Kossa, cells were washed with PBS and fixed with 95 % ice cold ethanol. For calcium retention, the fixed cells were exposed to 60 W UV light in a solution of 5 % silver nitrate for 60 min for visualization. Alkaline phosphatase (Alp) is a marker of proliferating osteoblasts and is greatly enhanced during in vitro bone formation. In brief, MC3T3-E1 and LNCaP-19 cells were fixed for 60 s using neutral buffered formalin (10 %) and washed with PBS + 0, 05 TWEEN 20. BCIP/NBT  substrate solution was added to the monolayer and incubated in dark for 25 min and washed before visualization. For the mineralization assays, medium was changed every 3 days.MC3T3-E1, LNCaP and LNCaP-19 cells were seeded in six well plates at a density of 1 × 10Runx2) , osterix (Osx) (Mm00501584_m1), msh homeobox 2 (Msx2) (Mm00442992_m1), alkaline phosphatase (Alp1) (Mm01187117_m1) osteocalcin (Ocn) , osteoprotegerin (Opg) (Mm01205928_m1) and endogenous control 18S rRNA (Hs99999901), purchased as TaqMan Gene Expression Assays (Applied Biosystems). PCR parameters were in accordance with the manufacturer’s protocol and the ΔΔCt method was used for relative mRNA quantification. The expression levels of each sample were normalized against 18S rRNA and PCR reactions for target genes, and controls were performed in triplicates for all samples.Total RNA was extracted using RNeasy Mini Plus kit  in accordance with the manufacturer’s instructions. RNA concentration was measured on a NanoDrop . A total of 1 μg RNA per reaction was reversely transcribed into cDNA using the VILO Superscript cDNA synthesis kit (Invitrogen) in accordance with the manufacturer’s instructions. RT-qPCR was performed using an ABI Prism 7500 Fast Sequence Detector  and specific TaqMan probes as follows: mouse runt-related transcription factor 2 (® Array Gene Signature Plate #4418741). The array included 96 osteogenesis-related genes and endogenous control genes . The array was used to evaluate the basal osteogenic expression profile of LNCaP-19, LNCaP, C4-2B4 and PC-3 with or without stimulation with OCM. Briefly, cells were seeded in T25 flasks in an optimized density  to reach 80 % confluence after 24 h. Cells were starved for 24 h before medium was replaced with control medium or OCM supplemented with 1 % FBS-DCC. Cultures were maintained for 48 h before RNA extraction, as previously described. A total of 5 μg cDNA/reaction was used for the PCR reactions. Parameters were set up in accordance with the manufacturer’s protocol and the ΔΔCt method was used for relative mRNA quantification. The expression levels of each sample were normalized against HPRT1 (Hs99999909_m1) and GUSB (Hs99999908_m1). Only target genes with the most significant variations are shown (twofold regulation). Genes were considered as non-detected in cases where Ct values were above 36. Gene expression analysis was performed in independent triplicates.Expression of bone-related genes in LNCaP-19, LNCaP and PC-3 were evaluated using a human osteogenesis gene signature array  were castrated via scrotal incision under anaesthesia. LNCaP-19 cells were then injected into the left tibia using a procedure similar to what has previously been described , 27. BriEthics statement: This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the local Committee on the Ethics of Animal Experiments of Gothenburg (Permit Number: 109-2008). All surgery was performed under anaesthesia, and all efforts were made to minimize suffering.Serum levels of Ocn, a marker of osteoblast activity and Opg, an inhibitor of osteoclast activity, were measured using mouse-specific ELISAs in accordance with the manufacturer’s instructions . Serum was diluted 1:10 and absorbance was read at 450 nm in a Wallac 1420 Victor3™ multiplate reader .t Test or Mann–Whitney U Test where appropriate. Statistical significance of BMD was determined by Mann–Whitney U test. Correlation was determined with Spearman’s rank correlation test. All data are presented as mean ± SEM. A P value of < 0.05 was considered significant.Statistical calculations were performed using the SPSSv 20 software package . Statistical differences between groups were performed using Student’s


The assay of this specific mRNA can be used to indicate the presence of live, as opposed to dead, cells of Salmonella enterica in a food matrix.The isolation of DNA-free RNA is a crucial step in the reverse transcription PCR (RT-PCR). Every RNA extraction procedure results in RNA samples contaminated with genomic DNA, which can cause false-positive outcomes in highly sensitive applications, including a recently developed quantitative real-time PCR (RT-qPCR) assay that targets Salmonella cells. The acceptability of the preparations for use in downstream applications such as RT-qPCR was judged in terms of the total amount of RNA recovered, the integrity of the RNA molecules, and minimal content of DNA. The five kits produced RNA preparations that differed markedly in yield, integrity of the Salmonella RNA and the amount of contaminant DNA. The greatest RNA recovery was achieved with the MasterPure kit; however, the preparation contained high levels of genomic DNA. The UltraClean extraction kit gave a low level of RNA recovery with a poor level of integrity. The RNeasy Mini, RiboPure and PureLink extraction kits produced high-quality, DNA-free RNA suitable for Salmonella detection by RT-qPCR.We evaluated the ability of five RNA extraction kits to produce RNA preparations from exponentially growing Salmonella invA mRNA by RT-qPCR. The use of these two kits will greatly reduce the frequency of false-positive results and might allow fast RT-qPCR determination of invA mRNA produced by viable Salmonella in food samples.We showed that the RNeasy Mini and PureLink RNA extraction kits were the most suitable for the detection of  Commercially available RNA extraction kits are rapid, capable of high-throughput analysis and cost-effective . The isoA260). The purity of the RNA sample can be determined using the A260/A280 ratio as a reference . However, the accuracy of this method is questionable, because protein contamination can cause an overestimation (>50%) of RNA content [The RNA concentration of a sample is commonly determined via measurement of absorbance at a wavelength of 260 nm  and Experion (Bio-Rad Laboratories) are widely used to estimate RNA quality and quantity [There are methods that allow for the accurate estimation of RNA concentration. Modern spectrometric methods, such as spectrophotofluorimetry , in combination with RNA RiboGreen dye  can be used for ultrasensitive RNA quantification quantity . Agilentquantity . RIN valquantity .Salmonella SE5 as a model organism. The kits evaluated were: the RiboPure Bacteria Kit (Ambion), the PureLink RNA Mini Kit (Invitrogen), the UltraClean Microbial RNA Isolation Kit , the RNeasy Mini Kit (Qiagen) and the MasterPure RNA Purification Kit (EPICENTRE). The kits were compared for (1) the yield of RNA, (2) the polymeric length integrity of the RNA and (3) the amount of DNA present in the RNA preparation.The present study evaluated the performance of five commonly used commercial RNA extraction kits for isolating cellular RNA, using actively growing Salmonella enterica serovar Enteritidis strain SE5 was grown overnight in Luria-Bertani (LB) medium_at 35°C with shaking (250 rpm).The performance of five commercial RNA extraction kits was evaluated: RiboPure-Bacteria Kit , PureLink RNA Mini Kit (Invitrogen), UltraClean Microbial RNA Isolation Kit (MoBio), RNeasy Mini Kit (QIAGEN) and MasterPure RNA Purification Kit (EPICENTRE Biotechnologies). The principles for RNA purification of these kits are very similar. The UltraClean and the RiboPure kits use a bead cell disruption system. Four of the kits, except the MasterPure kit, use spin-column technology and selective binding properties of silica membranes. The RNA adsorbs to the silica membrane in the presence of high concentrations of salt. Contaminants are unable to bind to the silica column and therefore pass through the column. The loaded column is washed and then any bound RNA is eluted. The MasterPure kit uses a salt-precipitation protocol, instead of a column, to purify the RNA and it captures the small RNA molecules that tend to be lost when using columns. All RNA extractions were done in triplicate (1 ml each) with exponentially growing SE5 cells and following the manufacturer's recommendations for each kit. The final RNA fraction was obtained by elution or suspended in 50 μl of DEPC-treated water (Ambion). The treatment with DNase I (Invitrogen) was done at 37°C for 30 min and the DNase was inactivated by incubation at 65°C for 10 min.The RNA concentration in individual RNA samples was determined using the RNA Pico 6000 LabChip kit (Agilent Technologies). The LabChips were run in an Agilent 2100 Bioanalyzer following the manufacturer's instructions (Agilent). The use of the RNA Pico 6000 LabChip kit allowed determination of the RNA integrity number (RIN), an indicator of the integrity of the RNA in the sample, using 2100 Bioanalyzer Expert software .invA mRNA levels were measured by RT-qPCR as described [invA RT-qPCR reactions were done as described [2 added to the master mix to a final concentration of 5 mM. The final concentration of each primer was 200 nM: (invA_176F 5'-CAACGTTTCCTGCGGTACTGT-3') and invA_291R (5'-CCCGAACGTGGCGATAATT-3'). The final concentration of the probe invA_Tx_208 was 150 nM (5'-TX-CTCTTTCGTCTGGCATTATCGATCAGTACCA-BHQ2-3'). RT-qPCR and data analysis (in triplicate) were done with a Rotor-Gene 3000 (Corbett) real-time PCR instrument using 2 μl of each RNA sample. Additionally, each RNA sample was amplified by invA qPCR to detect DNA contamination and to estimate the number of invA DNA copies present in the sample. The RT-qPCR conditions were as follows: 15 min at 50°C for the generation of the cDNAs, 2 min at 95°C to activate the hot-start Taq polymerase and then 40 cycles of denaturation at 95°C for 15 s, and primer annealing and extension at 60°C for 30 s (the acquisition of Texas Red emission was performed at the end of this cycle). Identical conditions were used for qPCR reactions. The reported efficiency of the qPCR and RT-qPCR for these primers is 0.93 - 0.99 and 0.90 - 0.96, respectively [https://www.genomics.agilent.com/CalculatorPopupWindow.aspx?CalID=8. For Cq values that dipped below this range, the RNA sample was considered inadequate for the detection of Salmonella invA mRNA.The escribed . The invescribed , but witTotal RNA concentrations were determined with a 2100 Bioanalyzer and the RNA 6000 Pico LabChip kit (Agilent) [The PureLink and the RNeasy kits use chemical methods to lyse the cells, whereas the RiboPure and UltraClean kits physically disrupt the bacterial cell walls by beating the cells with beads. Greater disruption would be expected to improve the RNA yield; however, the bead-based kits yielded less RNA than the others. This suggests that the different RNA yields of these kits are not due only to different extents of cell lysis.Salmonella RNA, as indicated by the RIN values of 8 or higher  results indicate the presence of DNA in the RNA samples [RT-PCR and RT-qPCR are important tools for the detection of pathogens in foods ,9-11. Thlmonella . An RT-q samples .invA mRNA RT-qPCR and invA qPCR results are given in Tables invA mRNA  and not to the quality of the RNA sample, which has been observed by others for different types of RNA samples [invA mRNA molecules in that sample ought to be smaller and could explain the 4 - 11-fold fewer copies observed for this kit compared to the others , which is unsuitable for some RNA assays. As expected, the treatment of RNA samples with DNase I after extraction appears to be absolutely required to reduce the amount of residual DNA, especially for sensitive assays like invA RT-qPCR. The RNeasy and RiboPure extraction kits produced large yields of RNA with a high degree of integrity and could be considered the kits of choice for the detection of Salmonella invA mRNA by RT-qPCR.Overall, this comparison showed that most of the kits tested were suitable for The authors declare that they have no competing interests.LVR did most of the RNA extractions, participated in the experimental design, and production of the draft of the manuscript. BA did some RNA extractions; participated in the experimental design, and production of the draft of the manuscript. NGE conceived the study, participated in the experimental design and coordination, did the RT-qPCR assays, and participated in production of the draft of the manuscript. All authors have read and approved the final manuscript.


The dominant theories about risky decision-making assume that decision conflicts are solved by a compensatory process involving a trade-off of probability against payoff, but it is unclear whether these theories actually represent the events that occur when people make a risky decision. By contrasting a preferential choice with a judgment-based choice that required a compensatory process, we explored the mechanisms underlying risky decision-making. First, using parametric analyses, we identified the dorsomedial prefrontal cortex (dMPFC) as the specific region in charge of task-related conflict in risky decision-making tasks. We also showed that the dMPFC was activated less when judgment-based choices were being made, implying that the conflict experienced during a judgment-based choice was not as strong as the conflict that was experienced during the preferential choice. Our results provide neural evidence that preferential choice cannot be characterized solely as a compensatory process. Thus, questions were raised about whether existing compensatory theories could adequately describe individual risky decisions. This idea seems simple and straightforward because decision-makers are only required to compute the mathematical expectation of each alternative with respect to function f before choosing the option that maximizes overall value or utility.Risky decisions become difficult when the payoff and probability are in conflict. Mainstream theories of decision-making under risk, from expected utility theory This assumption has led neuroscientists to attempt to identify the neural basis for each of the components that are required to produce an overall value or utility. Neuroimaging studies have identified potential neural substrates associated with payoff and probability However, it remains unclear whether the compensatory model actually represents what happens when people make a risky decision. The processes of weighing and summing have been challenged before e.g., . ResearcPsychological Review published a series of papers on this issue The debate about whether risky choices are based on a compensatory process or a non-compensatory process has a long history. Recently, the i.e., payoff and probability).The primary difference between compensatory processes and non-compensatory processes appears to be associated with the differences in the solutions to conflicts between binary alternatives Because few behavioral indices for monitoring internal conflict have been described Using the two factors  as the parameters for separate parametric analyses, we identified the conflict-related brain region in our study of risky decision making. Then, we showed differential neural sensitivities to these parameters between the preferential choice and the judgment-based choice. Finally, we showed evidence that the intensity of inner conflict revealed by our identified conflict-related region was less pronounced in the compensatory choice  than in the preferential choice. These neural measurements will help us to better understand the processes that underlie compensatory and non-compensatory decision making and to address the question of whether risky choices are guided by a compensatory process as assumed by expected utility theory and its extensions.Twenty-six undergraduate or graduate students  participated in this study. Three participants were excluded from the analyses because of excessive head motion. All participants were in good health with no previous history of psychiatric or neurological disease, and they gave written informed consent. The study was approved by both the Institutional Review Board of the Institute of Psychology, the Chinese Academy of Sciences, and the Institutional Review Board of the Beijing MRI Center for Brain Research.We constructed 60 pairs of two-outcome monetary bets: one featuring a high probability of winning/losing a modest amount of money (the P bet), and the other featuring a low probability of winning/losing a relatively large amount of money (the $ bet) . OutcomeIn the preferential choice task, participants were asked to select their preferred bet from each pair. To make the participants perform a compensatory process, we developed a judgment-based choice task using the certainty equivalent method Each participant performed both the preferential and judgment-based choice tasks with an interval of at least 7 days between the two tasks. Prior to entering the scanner, the participants played a practice version of either the preferential choice task or the judgment-based choice task to minimize learning effects during the actual scanning and to enable them to fully understand the paradigm. During the scanning, the participants were asked to perform the tasks with no time constraints. They made their decisions by pressing one of two buttons corresponding to the location of the bets on the screen. After the participants' response, there was a delay of 8 s before a 2 s fixation procedure started and indicated the next bets . There w2; slice thickness = 3 mm; slice gap = 1 mm and, thus, acquisition voxel size = 3.4×3.4×4 mm3). Each brain volume was composed of 32 axial sections. Stimuli were presented with E-prime software  on a personal computer, back-projected onto a screen using a liquid crystal display projector and viewed by participants through a mirror mounted on the MRI head coil. We also acquired a brief (6-min) resting-state scan, which was composed of 180 volumes (the data were not used in this study).MR images sensitized to changes in BOLD signal levels were obtained by an echo planar imaging sequence on a 3.0-Tesla Siemens MR scanner  analysis.Image preprocessing and analyses were performed using statistical parametric mapping  that was run on a MATLAB 7 platform . The first three volumes in each scan series, which were collected before equilibrium magnetization was reached, were discarded. The remaining images were corrected for within-scan acquisition time differences between the slices and then realigned to the first volume to correct for inter-scan head motions. Based on a visual inspection of the motion correction estimates, three participants who had more than 2-mm maximum displacement in any of the x, y or z directions or more than 2° of angular rotation about any axis for multiple volumes were excluded from this study. An additional two participants showed sudden head motion only in the first trial, which allowed the use of the imaging data obtained from these two participants after the first trial was removed. The realigned images were spatially normalized to the standard EPI template, resampled to 3×3×3 mmIndividual participant data were analyzed using the general linear model (GLM) with separate models for each task. Events were modeled with a variable-duration boxcar function convolved with a canonical hemodynamic response function (HRF). To quantitatively describe the relationship between brain activation and decision parameters, parametric analyses were performed.r = 0.483, p = 0.007) between the differences on the payoff dimension and the differences on the probability dimension. Thus, an increase in the difference in the payoff dimension would result in an increase in the difference in the probability dimension. To avoid the problems induced by severe co-linearity, we chose one intradimensional difference to enter into the regressor model at a time. Therefore, there were two parametric analyses for each task. For example, Model I was analyzed for the intra-dimensional difference in the probability dimension and the unidimensional difference, and Model II was analyzed for the intradimensional difference in the payoff dimension and the unidimensional difference.Trials were modeled with parametric regressor modeling (in the following order): (1) the intradimensional difference in the probability dimension or in the payoff dimension, and (2) the unidimensional difference between overall values/utilities of two bets on the unidimension alone. To avoid a choice in which one bet dominated the other, all of the bets used in our study were designed to be choices between high payoffs with low probabilities and low payoffs with high probabilities. As a consequence, we observed a significant correlation . By using the intradimensional difference in the probability/payoff dimension and the unidimensional difference between the cumulative prospect utilities of two bets as parameters, parametric analyses were conducted to identify brain regions that were sensitive to intradimensional difference and unidimensional difference. Paired t-tests were used to further verify the differences in the sensitivity to the specific decision parameters . To control for the difference in task difficulty between the two tasks, the average RT of each participant within each task was taken as an index of task difficulty and entered into the paired t-test analysis as a covariate.First-level (single participant) contrasts were performed separately over the mean trial activity (relative to baseline) and for each of the parametric regressors described above. Second-level (group) random effect analyses were completed by performing one-sample t-tests over each of these contrasts. Corrections for multiple comparisons were carried out at the cluster level using Gaussian random field theory implemented in SPM5 (min T(22)>2.5; cluster significance: p<0.05, corrected). Thus, a mask was formed in which each voxel value was set to one if the voxel satisfied one of the above criteria. The mask was set to zero if the criteria were not satisfied. To compare activities (relative to baseline) in the conflict-related regions between the two tasks, another random-effect paired t-test analysis controlling for the effect of task difficulty was used to determine where the second-level group contrasts differed between the preferential choice task and the judgment-based choice task. This analysis was limited to the mask .Finally, a mask composed of the conflict-related regions that were sensitive to the unidimensional difference and the intradimensional difference in the payoff or probability dimension was generated. According to our hypothesis, the conflict-related regions should be those regions in which the unidimensional difference between the two offered bets shows a negative modulation effect or regions in which the intradimensional difference on the probability/payoff dimension shows a positive modulation effect (min T(22)>2.5; cluster significance: F = 1.29, p>0.05) or domain  = 0.031, p>0.05). The interaction between task and domain was statistically significant  = 4.75, p<0.05). A simple effect test indicated that in the loss domain, participants selected more $ bets in the preferential choice task than in the judgment-based choice task (p<0.05).Behavioral results are shown in F = 10.90, p<0.01). They also took more time to make a decision in the loss domain than in the gain domain  = 7.77, p<0.05). The interaction between task and domain was not significant  = 2.32, p>0.05).We also conducted an ANOVA to compare the reaction time difference between the two tasks. Analyses of reaction time revealed that the participants took more time to make a judgment-based choice than to make a preferential choice  in two tasks. In the first block, the intradimensional difference in the probability dimension and in the payoff dimension were entered into the analysis using the enter method. In the second block, unidimensional differences between overall values/utilities of two bets were included using the stepwise method because it can identify those factor(s) for which the significance level is less than 5%. The included unidimensional differences between the two bets were derived from expected value (EV) theory, expected utility (EU) theory (where U = ln(x) or U = lg(x)), and the cumulative prospect theory .t = −2.019, standardized beta = −0.371, p<0.05), implying that a larger difference in the payoff dimension could effectively induce a smaller proportion selecting difference between the two bets . There were no other significant effects .For the preferential choice task, all of the unidimensional differences between overall values/utilities of two bets were excluded as predictors from the final regression model. There was an independent negative relationship between the intradimensional difference in the payoff dimension and the proportion selecting difference between the two bets , while the unidimensional differences between expected utilities (where U = ln(x) or U = lg(x)) were excluded as predictors from the final regression model. In the final model, the unidimensional difference in cumulative prospect utilities   and in expected values  showed positive relationships with the proportion selecting difference, while the unidimensional difference in cumulative prospect utilities  showed a negative relationship with the proportion selecting difference . The fact that the standardized beta coefficient of cumulative prospect utilities  was the largest positive coefficient suggests that the greater unidimensional difference  between the two bets could effectively induce a greater proportion selecting difference between the two bets .For the judgment-based choice task, inclusion of the unidimensional differences in expected values, cumulative prospect utilities , and cumulative prospect utilities  in the second block produced a significant F-change    , a positive correlation with the intradimensional difference was observed in the medial prefrontal cortex (MPFC) that extended to the anterior cingulate cortex (ACC)., and no opposite effects were observed in the preferential choice task. This positive modulation effect meant that the activity of the region increased when the difference in the probability dimension increased, just as we hypothesized. However, in the judgment-based choice task, no regions were sensitive to the intradimensional difference in the probability dimension (min T(22)>2.5; cluster significance: rrected) . A negatrrected) . Paired rrected)  and incrrrected) .p<0.05, corrected). However, in the judgment-based choice task, no regions were sensitive to the intradimensional difference in the payoff dimension (min T(22)>2.5; cluster significance: p<0.05, corrected)    and the MPFC (which survived the height but not the extent threshold) in the judgment-based choice task but not in the preferential choice task. Although the pattern in sensitivity to the unidimensional difference within each task was similar to the sensitivity in Model I, the differences between the two tasks in this model (Model II) were not significant in the MPFC even under an uncorrected threshold of p<0.01.Similarly, in the parametric regressor model with the intradimensional difference in the payoff dimension and the unidimensional difference between the cumulative prospect utilities of two bets as parameters (Model II), a positive correlation with the intradimensional difference in the payoff dimension was observed in the MPFC in the preferential choice task (min T(22)>2.5; cluster significance: rrected) . A paire = 3.89) . A negatp<0.005, uncorrected)  in the conflict-related regions between the two tasks to test whether the intensity of inner conflict was less pronounced in the judgment-based choice than in the preferential choice. Based on the parametric analyses, the conflict-related regions that were sensitive to the unidimensional difference and the intradimensional difference in the probability/payoff dimension were obtained. Because the BOLD activities (relative to baseline) in the Model I were exactly the same as the activities in Model II and because both models were based on the same task regressor, we only showed the results from Model I. Using Model I, the dMPFC showed increased activity in the preferential choice task compared to the judgment-based choice task (rrected) . No regiUnderstanding the underlying mechanism of decision making under risk remains a fundamental and difficult problem. Although there has been much debate about whether risky choice is based on compensatory or non-compensatory processes, to our knowledge, nothing has previously emerged to give decision analyst a pause about using the compensatory rule as the fundamental principle of decision making. The family of compensatory theories has kept its dominant position, in part through continuing generations of prospect theory The choice result revealed that our participants tended to choose one member of the pair but set a higher certainty equivalent for the other pair. However, the observations made about preferential choice could still be interpreted by compensatory proponents as being guided by a compensatory rule. For instance, the contingent weighting model Previous research also reported that compensatory processing takes more time than non-compensatory processing A novel behavioral finding was that the intradimensional difference and the unidimensional difference influenced preferential choice and judgment-based choice, respectively, in terms of the absolute difference between the proportion of participants selecting the $ bet and the proportion of participants selecting the P bet in each pair. Such a finding suggests that the impact of intradimensional versus unidimensional differences between two bets is task dependent (task specific). Thus, preferential choice is influenced by intradimensional differences but not by unidimensional differences between two bets, whereas judgment-based choice is influenced by unidimensional differences but not by intradimensional differences between two bets. Notably, an even selection (50:50) without strong preference for either bet indicates that participants were unable to detect a dominance relationship between the bets and thus experienced great conflict.Cognitive neuroimaging and neuropsychology studies have consistently shown that the ACC/dMPFC, which has been widely interpreted to respond to the presence of conflict Considering that the functional role of the ACC/dMPFC might be context-specific As our interpretations were made under the very particular conditions of risky decision making tasks, this interpretation should be regarded as tentative until further work excludes alternative interpretations.Guided by the different theoretical orientations, researchers have proposed different possible resolutions to conflict from the compensatory and non-compensatory points of view. In the debate between the compensatory and non-compensatory models, various behavioral methods, ranging from off-line questionnaires When there was a choice available between high payoffs with low probabilities and low payoffs with high probabilities, the participants seemed to employ a less mathematically complex strategy to make decisions in the preferential choice situation because these decisions were associated with faster response time. More importantly, the hierarchical multiple regression analyses suggested that participants used different strategies to make decision in two tasks. In the preferential choice task, increased proportion selecting difference was associated with a decrease in the difference in the payoff dimension, implying that the conflict pattern (in terms of the proportion selecting difference) in the preferential choice resulted from using a non-compensatory process without trading off probability against payoff. In the judgment-based choice task, on the other hand, increased proportion selecting difference was associated with an increase in the unidimensional difference (estimated using cumulative prospect theory), implying that the conflict pattern in the judgment-based choice was induced by the use of a compensatory process of trading off probability against payoff. Furthermore, we found a clear difference in the activation of the dMPFC between the responses to the two parameters in the two different tasks. The parametric analyses revealed that the parameters of the unidimensional difference (estimated using cumulative prospect theory) were poor predictors of dMPFC activation in the preferential choice but were good predictors of dMPFC activation in the judgment-based choice. In contrast, the parameters of the intradimensional difference (payoff and probability separately) were good predictors of the dMPFC activation in the preferential choice, but they were poor predictors of dMPFC activation in judgment-based choice. These results provided behavioral and neural evidence that preferential choice is non-compensatory, or at least not purely compensatory.To the extent that dMPFC activity has been identified as playing an important role in conflict monitoring, our finding that dMPFC activation increased during the preferential choice task suggests that the conflict participants experienced during the judgment-based choice was not as strong as the conflict experienced during the preferential choice. This finding strengthens our doubt that individuals were seeking a dominant alternative as a means of solving decision conflict by using the weighing and summing process that was suggested by a compensatory rule.A limitation of our study is that we only completed a global check rather than a trial-by-trial check by asking participants to explicitly indicate the certainty equivalence for each bet during scanning. There is a possibility that participants' certainty equivalence estimate may vary for repeated attempts to estimate the value of a gamble in the judgment-based choice task. Although the longer RT and the positive relationship between the proportion selecting difference and the unidimensional difference in the judgment-based choice task provide indirect evidence that participants tend to follow the certainty equivalent method when making a judgment-based choice, further research should continue to examine this possibility.In summary, by examining brain activity, we supported our hypothesis that preferential choice cannot be characterized as a compensatory process with the aim of maximizing the overall value of a prospect in a conflict solution. Rather, it seems that preferential choice may be a non-compensatory process that is accompanied by strong conflicts. Together, these results tell a simple story: selection of the bet with the greatest overall value may appear to be an ideal way to solve conflict, but it does not capture the nature of risky decision making. Our study, from a perspective of decision conflict, attempted to address the issue of whether risky choices are based on a compensatory process. This perspective could potentially lead to a better understanding of the underlying mechanism of risky decision-making, which could present important directions for future research.


Here we visualize the complex muscle-specific processing pattern of the unc-60 pre–mRNA with asymmetric fluorescence reporter minigenes. By disrupting juxtaposed CUAAC repeats and UGUGUG stretch in intron 1A, we demonstrate that these elements are required for retaining intron 1A, as well as for switching the processing patterns of the entire pre–mRNA from non-muscle-type to muscle-type. Mutations in genes encoding muscle-specific RNA–binding proteins ASD-2 and SUP-12 turned the colour of the unc-60 reporter worms. ASD-2 and SUP-12 proteins specifically and cooperatively bind to CUAAC repeats and UGUGUG stretch in intron 1A, respectively, to form a ternary complex in vitro. Immunohistochemical staining and RT–PCR analyses demonstrate that ASD-2 and SUP-12 are also required for switching the processing patterns of the endogenous unc-60 pre-mRNA from UNC-60A to UNC-60B in muscles. Furthermore, systematic analyses of partially spliced RNAs reveal the actual orders of intron removal for distinct mRNA isoforms. Taken together, our results demonstrate that muscle-specific splicing factors ASD-2 and SUP-12 cooperatively promote muscle-specific processing of the unc-60 gene, and provide insight into the mechanisms of complex pre-mRNA processing; combinatorial regulation of a single splice site by two tissue-specific splicing regulators determines the binary fate of the entire transcript.Pre–mRNAs are often processed in complex patterns in tissue-specific manners to produce a variety of protein isoforms from single genes. However, mechanisms orchestrating the processing of the entire transcript are not well understood. Muscle-specific alternative pre–mRNA processing of the  unc-60 gene locus, encoding two tissue-specific isoforms of ADF/cofilin in C. elegans. The unc-60A and unc-60B genes share only the first exon, and UNC-60B protein is specifically expressed in muscle. We visualize the tissue-specific processing patterns of the unc-60 pre–mRNA with green and red fluorescent proteins in living worms. We provide genetic, biochemical, and immunohistochemical evidence that muscle-specific RNA–binding proteins ASD-2 and SUP-12 cooperatively bind to specific motifs in intron 1A to retain intron 1A, which leads to skipping of exon 2A through 5A and splicing between exon 1 and 2B. Consistently, disruption of the splicing factors leads to expression of UNC-60A in muscle and suppresses paralysis of an unc-60B-specific mutant. Our study raises a model of step-by-step execution of complex co-transcriptional pre–mRNA processing and provides insight into the fate decision of the entire transcript.Muscle is a specialized organ with specialized contractile apparatuses. A number of genes encoding contractile apparatus-related proteins undergo muscle-specific pre–mRNA processing. However, the molecular mechanisms and consequences of muscle-specific alternative pre–mRNA processing remain largely unknown. In this study, we reveal regulation mechanisms of pre–mRNA processing of the  These include muscleblind-like (MBNL) Muscle is one of tissues in which many genes undergo tissue-specific pre-mRNA processing uncoordinated (unc)-60 gene in Caenorhabditis elegans provides an excellent model of muscle-specific and complex pre-mRNA processing of genes related to contractile apparatuses. The unc-60 gene encodes two homologous proteins, UNC-60A and UNC-60B unc-60 gene consists of a common first exon and two separate series of downstream exons, exons 2A through 5A for UNC-60A and exons 2B through 5B for UNC-60B , is required for generation of muscle-specific UNC-60B mRNA unc-60 gene remains unclear. In this study, we applied a transgenic alternative splicing reporter system unc-60 pre-mRNA. We demonstrate that repression of excision of the intron between exon 1 and exon 2A is the fate-determining event for the unc-60 transcript. We provide genetic and biochemical evidence that SUP-12 and another muscle-specific splicing regulator Alternative-Splicing-Defective-2 (ASD-2), a member of the signal transduction and activation of RNA (STAR) family of RNA-binding proteins in vivo evidence that combinatorial regulation of a single splice site by two tissue-specific splicing regulators determine the binary fate of the entire transcript that can potentially be processed into two alternative isoforms.The structure of the unc-60 transcript in vivo, we intended to construct a pair of fluorescence alternative processing reporter minigenes. If the intron between exon 1 and exon 2A  is excised prior to selection of exon 2B, it would be impossible to produce UNC-60B mRNA. We therefore assumed that excision of intron 1A should be repressed until exon 2B is transcribed in tissues where UNC-60B is expressed. On the basis of the assumption, we constructed an asymmetric pair of reporter minigenes, unc-60E1-E2A-RFP and unc-60E1-E3B-GFP. The unc-60E1-E2A-RFP cassette, carrying unc-60 genomic DNA fragment from exon 1 through exon 2A  products, and confirmed that the four mRNA isoforms schematically shown in We successfully visualized the alternative expression of the UNC-60 isoforms with the unc-60 processing, we utilized myo-3 promoter to drive expression of the unc-60 reporter specifically in body wall muscles. Transgenic worms with an integrated transgene allele ybIs1831 [myo-3::unc-60E1-E2A-RFP myo-3::unc-60E1-E3B-GFP] predominantly expressed UNC-60B-GFP in body wall muscles sup-12 background -15egl-15 exon 5B, SUP-12 functions as a muscle-specific partner of the Fox-1 family proteins ASD-1 and FOX-1 unc-60. As direct interaction between SUP-12 and ASD-1 in a yeast two-hybrid system had been reported in a worm interactome study unc-60 reporter by knocking down genes encoding possible SUP-12-interactors ASD-1, ASD-2, ETR-1, MEC-8, R02F2.5 and W02A11.3, deposited in the database (http://interactome.dfci.harvard.edu/). We performed RNA interference (RNAi) by feeding the reporter worms with bacterial clones targeting the six genes, and found that knockdown of asd-2 led to expression of UNC-60A-RFP  has a nonsense mutation in the asd-2b-specific first exon (unc-60 reporter worms exhibited weak Red phenotype in the asd-2 (yb1540) background  worms with a purified immunoglobulin G (IgG) fraction (asd-2 mutant (asd-2 (yb1540) lysate (asd-2 (yb1540) mutant.We previously identified ASD-2, an RNA-binding protein belonging to the STAR family, as a regulator of muscle-specific and developmentally regulated alternative splicing of a collagen gene rst exon , which ickground  and bodyckground , confirmfraction . Nuclei fraction  and not 2 mutant . In West) lysate . These r) lysate , suggestunc-60 reporter minigenes in body wall muscles, we performed RT-PCR analysis with minigene-specific primer sets  revealed that CTAAC repeats and TGTGTG stretch are highly conserved just upstream of the splice acceptor site  worms  worms . RT-PCR minigene , indicatcis-elements in unc-60 intron 1A in vitro, we prepared radiolabelled RNA probes containing the intact sequence (WT) or those with mutations as in the mutant reporters (M1 and M2) (To confirm direct and specific binding of ASD-2 and SUP-12 to the  and M2)  and reco and M2)  to perfo and M2) . Recombiunc-60 intron 1A RNA. We analyzed supershifts of the mobility of the unc-60 intron 1A probes by the combination of ASD-2b and SUP-12 in EMSAs (unc-60 intron 1A RNA. ASD-2b failed to supershift the mobility of M1 probe (We next asked whether ASD-2b and SUP-12 cooperatively bind to in EMSAs . ASD-2b unc-60 intron 1A by pull-down experiments (S-transferase (GST)-fused full-length ASD-2b protein pulled down a substantial amount of recombinant full-length SUP-12 protein in the absence of target RNAs (unc-60 intron 1A (unc-60-I1A) RNA enhanced the pull-down efficiency in a dose-dependent manner . On the other hand, GST-fused monomeric RFP (mRFP) protein failed to pull down SUP-12 protein even in the presence of unc-60-I1A RNA (lanes 10–13), demonstrating specific interaction between ASD-2b and SUP-12. M1 and M2 mutant unc-60-I1A RNAs less effectively enhanced the interaction between ASD-2b and SUP-12 (lanes 5–8), consistent with their weaker or no ability to form a ternary complex ; asd-2 (RNAi) worms  worms . To furti) worms . In wildsup-12 mutation strongly suppressed structural defects of body wall muscles and paralysis of UNC-60B-specific mutant, unc-60B (su158)su158 lacks exons 3B and 4B  mutant. Wild-type worms exhibited sinusoidal locomotion (unc-60B (su158) worms were almost paralyzed (asd-2 (yb1540); unc-60B (su158) double mutant slightly restored motility and actin filament organization (asd-2(RNAi) worms showed a severer colour phenotype than asd-2(yb1540) allele ; unc-60B (su158); asd-2 (RNAi) worms restored sinusoidal locomotion (asd-2 (yb1540) mutation and/or asd-2 (RNAi) resulted in ectopic expression of UNC-60A in body wall muscles in the unc-60B (su158) background (unc-60B (su158) mutant, indicating that UNC-60A can exert, at least in part, functions of muscle-specific UNC-60B isoform and that possible splicing change in other genes are not required for the phenotype suppression. These observations demonstrate that ASD-2 is a bona fide regulator of the muscle-specific pre-mRNA processing of the endogenous unc-60 gene as well as SUP-12.Our previous work demonstrated that B and 4B , and supnization . Since a) allele , we furtckground . Transgeckground  as well ckground  in body unc-60 gene (sup-12 (yb1253) worms because asd-2 (yb1540) mutation exhibited weaker effect on the unc-60 reporter. In the wild type, mature UNC-60A and UNC-60B mRNAs were almost equally detected (sup-12 mutant (lane 4), consistent with the result with the reporter (sup-12 mutant (lane 4), consistent with the result shown in sup-12 mutant, all the introns, including intron 1A, were excised in the only detected RNAs . These RNAs were all the theoretical intermediates in the UNC-60A processing. In the wild type (lane 3), two of the RNAs (bands 3 and 6) predominated, suggesting that SUP-12 represses their production. In these RNAs, intron 1A alone (band 6) or introns 1A and 2A were retained (band 3), supporting the idea that SUP-12 represses excision of intron 1A, and weakly of intron 2A, even after introns 3A and 4A are excised. We then analyzed the partially spliced RNAs with the forward primer in exon 1 and intronic reverse primers in introns 2A, 3A and 4A (sup-12 mutant (lane 4), and relative amounts of the partially spliced RNAs to the pre-mRNAs (band 1) in the wild type (lane 3) and sup-12 mutant (lane 4) were in good accordance with the idea that excision of introns 1A and 2A is facilitated in the absence of SUP-12. All these analyses of the partially spliced RNAs supported the model that SUP-12 represses excision of intron 1A to preserve exon 1 until exon 2B is transcribed in muscles.We next analyzed partially spliced RNAs from the UNC-60A region . AlthougA and 4A . All theunc-60 pre-mRNA. unc-60 transcript, although the results demonstrated above did not provide conclusive evidence that ASD-2 and/or SUP-12 regulate muscle-specific repression of the polyadenylation site for UNC-60A mRNA.In this study, we have provided genetic and biochemical analyses of the mechanisms for regulation of the muscle-specific alternative processing of the C. elegans does not have a recognizable branch point consensus or a polypyrimidine tract egl-15 exon 5B, where the Fox-1 family proteins and SUP-12 cooperatively bind to juxtaposed cis-elements overlapping a putative branch site sup-12 identified many other splicing events affected by multiple splicing factors C. elegans.We have demonstrated that ASD-2 and SUP-12 cooperatively represses the 3′-splice site and not the 5′-splice site of intron 1A. Although Drosophila, Held out wings (How) ASD-2 ortholog in unc-60 gene is the cholinergic gene locus; genes encoding choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT) share the common first exon, and the other exon(s) for VAChT reside in the first intron of the ChAT gene in mammals DrosophilaC. elegansIn this study, we have presented a model of complex alternative pre-mRNA processing of a gene generating two almost distinct mRNAs. An important aspect of this study is the successful application of a dichromatic fluorescence reporter system to analyze the complex alternative pre-mRNA processing. The asymmetric pair of fluorescence reporter minigenes utilized in this study offers an alternative option for visualizing complex processing patterns besides symmetric pairs of minigenes applied to mutually exclusive exons and cassette exons unc-60B mutant. However, both UNC-60A and UNC-60B have characteristic actin-regulatory activities of ADF/cofilin in vitro with some quantitative differences unc-60B mutant muscles still exhibit minor disorganization (unpublished data), suggesting that UNC-60B is a more suitable isoform. On the other hand, UNC-60B cannot compensate for the function of UNC-60A in the gonadal myoepithelial sheath We demonstrated that ectopically expressed UNC-60A can compensate for the function of UNC-60B in sarcomeric actin organization in body wall muscles of unc-60E1-E2A-RFP and unc-60E1-E3B-GFP cassettes, unc-60 genomic fragments spanning from exon 1 through 2A and exon 1 through 3B, respectively, were amplified from N2 genomic DNA and cloned into Gateway Entry vectors (Invitrogen) carrying either mRFP1 M1 and M2 mutations were introduced by mutagenesis with Quickchange II (Stratagene). Expression vectors were constructed by homologous recombination between the Entry vectors and Destination vectors To construct the lin-15 (n765) as a host or pmyo-2-mRFP as a marker. Integrant lines were generated by ultraviolet light irradiation as described previously Worms were cultured following standard methods. Transgenic lines were prepared essentially as described asd-2 RNAs were prepared as described preciously RNAi experiments by feeding were performed essentially as described Total RNAs were extracted from worms by using RNeasy Mini kit (Qiagen) and DNase I (Promega). RNAs (300–500 ng) were reverse transcribed using random hexamers and Superscript II (Invitrogen) according to manufacturer's protocol. PCR was performed essentially as described previously Denatured His-tagged full-length ASD-2b for immunization was purified from denatured bacterial lysate by using Ni-NTA agarose (QIAGEN). Cold-shock inducible expression vectors for His-GST-fused full-length ASD-2b and mRFP1 and FLAG-tagged full-length SUP-12 were constructed by using Destination vectors pDEST-Cold-GST and pDEST-Cold-FLAG (H.K.), respectively. GST-ASD-2b and FLAG-SUP-12 were purified by using Glutathione Sepharose 4B  and Anti-FLAG M2 Magnetic Beads (Sigma), respectively, and dialyzed against RNA binding buffer (see below). Purified proteins were separated by standard SDS-PAGE and stained with SimplyBlue SafeStain (Invitrogen).Rabbit polyclonal anti-ASD-2b antiserum was generated with denatured recombinant His-ASD-2b protein by Operon Biotechnologies . IgG fraction (TD0135-02) was prepared from the antiserum by Medical & Biological Laboratories . Worm lysates were extracted from synchronized L1 larvae, separated by neutral polyacrylamide gel electrophoresis  and transferred to nitrocellulose membrane . Western blotting was performed with 15 µg/ml anti-ASD-2b (TD0135-02) or 1∶40,000-diluted anti-actin monoclonal antibody  and 1∶1,000-diluted HRP-conjugated anti-rabbit IgG antibody (Pierce) or 1∶10,000-diluted HRP-conjugated anti-mouse IgM antibody . Chemiluminescence signals  were detected by using LAS4000 .asd-2 (yb1540) worms were fixed with Bouin's fixative  supplemented with 25% methanol and 1.25% 2-mercaptoethanol for 60 min at room temperature, washed with phosphate-buffered saline (PBS) and permeabilized with 5% 2-mercaptoethanol and 1% Triton X-100 in PBS at 37°C for 30 hours. Fixed worms were treated with blocking buffer  in PBS) for 2 hours at room temperature and stained with 6 µg/ml anti-ASD-2b (TD0135-02) as a primary antibody in blocking buffer for 24 hours at room temperature and then with 2 µg/ml Alexa488-conjugated goat anti-rabbit IgG (Invitrogen) as a secondary antibody together with 1 µg/ml Hoechst 33258 (Hoechst) in blocking buffer for 2 hours at room temperature. Fluorescence images were captured by using a compound microscope  equipped with a colour, cooled CCD camera  or an inverted fluorescence microscope (Nikon TE2000) equipped with a monochrome CCD camera . Staining with anti-UNC-60A and anti-MyoA were performed as described previously For staining with anti-ASD-2b, mixed stages of N2 and 32P-labelled RNA probes were generated by in vitro transcription with [α32P] UTP (Perkin Elmer) and T7 RNA polymerase (Takara). Sequences of template oligo DNAs are available in E. coli tRNA and 50 ng/µl BSA for 30 min at 20°C. Each sample was separated on a non-denaturing 4% polyacrylamide gel and analyzed with a fluoro-imaging analyzer .E. coli tRNA, 50 ng/µl BSA and 0, 30, 100, or 300 nM of unc-60-I1A RNAs (Operon Biotechnologies) for 30 min at 20°C. The sequences of the unc-60-I1A RNAs: unc-60-I1A-WT, 5′-UUUUUGCCUAACCUAACCUAACCUAUGUGUGCCUGUUUU-3′; unc-60-I1A-M1, AAACCAAACCAAACCUAUGUGUGCCUGUUUU-3′5′-UUUUUGCC; unc-60-I1-M2, AUAUACCUGUUUU-3′5′-UUUUUGCCUAACCUAACCUAACCUAU. Beads were washed four times with 1 ml pull-down buffer. Bound proteins were eluted with LDS sample buffer and separated by NuPAGE (Invitrogen). Gels were stained with SimplyBlue SafeStain (Invitrogen) and detected and analyzed by using LAS4000 .His-GST-fused recombinant full-length ASD-2b and mRFP1 proteins were immobilized on glutathione sepharose 4B beads  and incubated with His-SUP-12 in 100 µl of pull-down buffer (20 mM HEPES-KOH (pH7.9), 150 mM KCl, 1% Triton X-100, 1 mM DTT and 0.1 mM PMSF) supplemented with 100 ng/µl Figure S1unc-60 reporter expression. Microphotographs of ybIs1831 worms fed with bacterial clones expressing dsRNAs for indicated genes. Images in red channels are pseudo-coloured in magenta. Scale bar, 100 µm.RNAi knockdown of SUP-12-interacting proteins revealed ASD-2 as a candidate regulator of the (PDF)Click here for additional data file.Figure S2unc-60 mRNAs from synchronized L1 worms of N2 (lane 1), asd-2 (yb1540) (lane 2), asd-2 (yb1540); ybIs1831; control (RNAi) (lane 3) and asd-2 (yb1540); ybIs1831; asd-2 (RNAi) (lane 4). Splicing patterns of the mRNAs are schematically shown on the right. Triangles indicate positions and directions of the primers.RT–PCR analysis of the endogenous (PDF)Click here for additional data file.Figure S3unc-60 (su158) (A), unc-60 (su158); asd-2 (RNAi) (B), asd-2 (yb1540); unc-60 (su158) (C) and asd-2 (yb1540); unc-60 (su158); asd-2 (RNAi) (D) worms. MyoA is a marker for body wall muscles (encircled with dotted lines in left panels). Scale bar, 20 µm.Immunofluorescence images of UNC-60A (left) and MyoA (middle) and merged images (right) of (PDF)Click here for additional data file.Table S1Sequences of primers used in plasmid construction.(RTF)Click here for additional data file.Table S2Sequences of primers used in RT–PCR assays.(RTF)Click here for additional data file.Table S3in vitro transcription.Sequences of oligo DNAs used in (RTF)Click here for additional data file.


Ribonucleic acids (RNA) are generally considered fragile molecules that are readily degraded. However, there is growing documentation of long-term (from days to centuries) RNA persistence in a variety of contexts and tissue types, and as such a number of academic disciplines are beginning to exploit degraded RNA. While the reasons for its survival are not fully understood, there are several plausible mechanisms that would safeguard this molecule against degradation. However, after examining the literature available on the postmortem instability and decay mechanisms of RNA, it has become clear that limited experimental studies and no reviews offer an overview of these mechanisms. Hence in this review we outline molecular reasons for RNA surviving long-term postmortem, and provide specific examples of RNA survival in forensic, archival and archaeological contexts. A better understanding of the mechanisms of RNA decay will be crucial for developing expectations on its long-term survival. The potential of RNA as a source of genetic information remains relatively unexplored in postmortem studies. This is possibly due to the well-documented phenomena that in many common situations, RNA degrades more readily than DNA. However, RNA can provide us with valuable information not directly evident in the genome and hence could be worth examining further in postmortem samples. For instance, unlike the genome, RNA transcripts provide access to gene regulation and protein information . MoreoveMechanisms behind DNA degradation and its effects on ancient DNA (aDNA) were reviewed by Lindahl  in a papThe half-life of nucleic acids is limited by several endogenous  and exogenous factors . MoreWhile DNA fragments of quality that would enable conventional PCR analyses are estimated to survive at least 100,000 years at the colder extremes of the ambient temperatures naturally found on Earth , convent2+.In contrast to DNA, the RNA molecule contains a hydroxyl group (2′-OH) at the 2′ position of the sugar  [A further relevant feature is that RNA readily forms secondary and tertiary structures, and these can have a distinct effect on the rate and specificity of RNA phosphodiester bond hydrolysis. In particular, ribosomal RNA (rRNA) is more stable than messenger RNA (mRNA), most likely due to the ability of rRNA to be partially double-stranded and form secondary structures. The difference in RNA degradation rates of these two RNA species forms the basis of postmortem interval determination . Tertiary structures are further stabilized by ionic interactions, for instance MgA (tRNA) . Hence, Additionally, micro RNA (miRNA) is exceptionally stable postmortem, which may be attributed to its ability to bind to proteins and subcellular compartmentalization . MoreoveAlthough RNA in itself is a fragile molecule, there are several factors safeguarding it, and in some situations it may be expected to persist, thus opening up possibilities for exploitation in a range of fields. So far this has encompassed the forensic, medical and archaeological sciences.The potential utility of RNA for the field of forensic science is a recent concept ,15. The et al. [et al. [et al. [Postmortem RNA degradation has been explored in a variety of tissue types. For example, Marchuk et al.  indicate [et al.  also rep [et al.  has sign [et al. ,23. Thes [et al. ,23. Addi [et al.  and bone [et al.  have bee [et al.  report het al. [Although mRNA has been demonstrated to survive for extended periods of time, mRNA is nonetheless highly susceptible to decay, with the half-life of various mRNA molecules varying between several weeks, hours, or even minutes for specific induced genes . This diet al. ,31, for As an intermediate for protein synthesis, mRNA levels reflect tissue-specific gene expression, as different tissues require different levels of proteins for specific enhanced expression . This haet al. [It should also be noted that different mechanisms of degradation  suggest that different features of RNA species would determine their postmortem survival. For instance, it has been established that the RNA decay rate within living cells is associated with the presence of AU-rich motifs in the RNA sequence . Catts eet al.  demonstrThe examples above drawn from forensic sciences highlight that informative amounts of RNA can clearly survive postmortem, conditional on tissue type and the conditions at death/burial. It should, however, be emphasized that the majority of forensic RNA investigations have been carried out in controlled conditions with samples that have been kept in known storage conditions. Exposure to sunlight, humidity, high temperature and other unfavorable influences might either lead to total loss of extractable RNA or RNA fragmentation might be accelerated. Hence, based on the success of the age determination and sample type, and the extended persistence of RNA in various tissue types (dried blood being the most favorable), additional investigation into the long-term survival of RNA in both a laboratory setting and in genuine casework conditions is warranted.Formalin-fixed paraffin-embedded (FFPE) tissue samples are the most common form of pathologic tissue collection  and offeet al. [et al. [Improved extraction and amplification techniques have resulted in a proliferation of cancer-focused gene-expression studies using RNA from FFPE tissue, as previously shown ,47. Wholet al. . Ravo et [et al.  reportedFFPE archival material has also provided a means to retrospectively study infectious disease, providing a means of clarifying pathogen evolution and emergence. For example, two historically important pathogens have been studied through use of FFPE archival material, namely 1918 Spanish influenza and human immunodeficiency 1 virus (HIV-1) ,42.Genomic RNA from the 1918 influenza virus was recovered from archived formalin-fixed lung autopsy materials and from frozen, unfixed lung tissues from an Alaskan influenza victim who was buried in permafrost in November 1918 ,50,51. Ret al. [Similarly, Worobey et al.  reportedIn addition to providing insights into recent sample types such as forensic and medical samples, RNA has potential to contribute to the field of ancient biomolecules . It is iSilene stenophylla seed, it has become apparent that this is not the case [The primary tissue type that has received attention in the field of archaeological RNA is desiccated seeds. It has traditionally been thought that after prolonged storage, a seed loses its capacity to germinate. However, with the germination of a 2,000-year-old date palm and, more recently, the germination of a 30,000-year-old the case ,57. If tthe case .Lepidium sativum L.) seeds found in the Thebes Necropolis, dated approximately 1,400 years BC. These seeds were found to contain low molecular weight (10 bp) fragments of RNA, based on the detection of uracil using mass spectrometry analysis. Hybridization tests were performed to confirm that the RNA was of plant origin and not bacterial DNA. However, the possibility of modern contamination was not excluded. In 1990, Venanzi and Rollo [Cheah and Osborne  also demnd Rollo  claimed However, using second-generation sequencing, a recent study  has succin vivo is to be degraded, the extent of its survival postmortem is perhaps more than many would expect. The examples highlighted above not only provide evidence for RNA being more robust than previously considered, but also demonstrate the tissue types and environmental conditions in which one could expect RNA persistence. A common theme seen in all of the sample types  is that in order for RNA to be preserved, there needs to be some form of barrier or macromolecular structure between the environment and the nucleic acids. This is true for archaeological specimens (such as seed casing and virions) and for archival material (where the FFPE acts as the barrier). These barriers protect RNA from the two main sources of degradation, oxygen and water, as well as other environmental factors.Although the fate of RNA In conclusion, RNA can provide valuable information that is otherwise unobtainable through DNA analysis. As such, the long-term survival of RNA warrants further exploration and characterization in future studies.Bp: Base pairs; FFPE: Formalin-fixed paraffin-embedded; ADNA: Ancient DNA; PCR: Polymerase chain reaction; 2′-OH: 2 prime hydroxyl group; RT-PCR: Reverse transcriptase polymerase chain reaction; MRNA: Messenger RNA; MiRNA: MicroRNA; RRNA: Ribosomal RNA; TRNA: Transfer RNA.The authors declare no competing interests.SLF, MLK, NLVD and MTPG participated in the writing of the manuscript. All authors read and approved the final manuscript.


Salmo salar); a disease first diagnosed in Norway in 1984. This virus, which was first characterized following its isolation in cell culture in 1995, belongs to the family Orthomyxoviridae, genus, Isavirus. The Isavirus genome consists of eight single-stranded RNA segments of negative sense, each with one to three open reading frames flanked by 3' and 5' non-coding regions (NCRs). Although the terminal sequences of other members of the family Orthomyxoviridae such as Influenzavirus A have been extensively analyzed, those of Isavirus remain largely unknown, and the few reported are from different ISAV strains and on different ends of the different RNA segments. This paper describes a comprehensive analysis of the 3' and 5' end sequences of the eight RNA segments of ISAV of both European and North American genotypes, and evidence of quasispecies of ISAV based on sequence variation in the untranslated regions (UTRs) of transcripts.Infectious salmon anemia (ISA) virus (ISAV) is a pathogen of marine-farmed Atlantic salmon (Two different ISAV strains and two different RNA preparations were used in this study. ISAV strain ADL-PM 3205 ISAV-07 (ADL-ISAV-07) of European genotype was the source of total RNA extracted from ISAV-infected TO cells, which contained both viral mRNA and cRNA. ISAV strain NBISA01 of North American genotype was the source of vRNA extracted from purified virus. The NCRs of each segment were identified by sequencing cDNA prepared by three different methods, 5' RACE (Rapid amplification of cDNA ends), 3' RACE, and RNA ligation mediated PCR. Sequence analysis of five clones each derived from one RT-PCR product from each NCR of ISAV transcripts of segments 1 to 8 revealed significant heterogeneity among the clones of the same segment end, providing unequivocal evidence for presence of intra-segment ISAV quasispecies. Both RNA preparations (mRNA/cRNA and vRNA) yielded complementary sequence information, allowing the simultaneous identification and confirmation of the 3' and 5' NCR sequences of the 8 RNA genome segments of both genotypes of ISAV. The 3' sequences of the mRNA transcripts of ADL-ISAV-07 terminated 13-18 nucleotides from the full 3' terminus of cRNA, continuing as a poly(A) tail, which corresponded with the location of the polyadenylation signal. The lengths of the 3' and 5' NCRs of the vRNA were variable in the different genome segments, but the terminal 7 and 11 nucleotides of the 3' and 5' ends, respectively, were highly conserved among the eight genomic segments of ISAV. The first three nucleotides at the 3' end are GCU-3' (except in segment 5 with ACU-3'), whereas at the 5' end are 5'-AGU with the polyadenylation signal of 3-5 uridines 13-15 nucleotides downstream of the 5' end terminus of the vRNA. Exactly the same features were found in the respective complementary 5' and 3' end NCR sequences of the cRNA transcripts of ADL-ISAV-07, indicating that the terminal sequences of the 8 RNA genome segments are highly conserved among the two ISAV genotypes. The 5' NCR sequences of segments 1, 2, 3, 5, and 7, and the 3' NCR sequences of segments 3 and 4 cRNA were 100% identical in the two genotypes, and the 3' NCR sequences of segment 5 cRNA was the most divergent, with a sequence identity of 77.2%.T/ATTTTTACT-3' (in the message sense 5'-3') in all segments of both ISAV genotypes.We report for the first time, the presence of intra-segment ISAV quasispecies, based on sequence variation in the NCR sequences of transcripts. In addition, this is the first report of a comprehensive unambiguous analysis of the 3' and 5' NCR sequences of all 8 RNA genome segments from two strains of ISAV representing the two genotypes of ISAV. Because most ISAV sequences are of cDNA to mRNA, they do not contain the 3' end sequences, which are removed during polyadenylation of the mRNA transcripts. We report for the first time the ISAV consensus sequence CA Salmo salar); a disease first diagnosed in Norway in 1984 ,,Orthomyxsegments , and 8 [segments , indicatsegments  till nowsegments ,28 and 7segments ,33) are segments .337V to M372 in the stem of the HE protein, adjacent to the transmembrane region [Sequence analysis of several ISAV isolates in the ORFs on the eight genomic RNA segments consistently reveals two genotypes that are designated with respect to their geographic origin, European and North American; the two show 15-19% difference in their amino acid sequences of the surface glycoproteins, fusion (F) protein and haemagglutinin-esterase (HE) protein . It has e region , but thee region . There ie region ,15.et al. [ISAV being an orthomyxovirus is characterized by abundant genetic variation. Orthomyxoviruses such as influenza viruses have high mutation rates because the viral RNA-dependent RNA polymerases have a high misincorporation frequency and have no proofreading-repair mechanisms . Moreoveet al.  used seqet al. . To examet al. , it was The 5' and 3' UTR sequences of viral RNA segments were determined using the Rapid Amplification of cDNA ends (RACE) method. For performing the 3' RACE, total RNA containing viral mRNA and cRNA was reverse transcribed to cDNA, which was then amplified by PCR using the ISAV segment specific primers Table  and adapThe 5' RACE protocol with ISAV segment specific primers Table  did not In designing this study, it was considered that the diversity of terminal sequences in a population of viruses present in an infected cell lysate as represented by RT-PCR would be a true indication of ISAV quasispecies. For this, two types of viral transcripts were studied; viral mRNA transcripts were analyzed using the 3'RACE protocol whereas viral cRNA transcripts were analyzed using RNA ligation method Figure . Thus, aThe lengths of the 3' UTR sequences for mRNA transcripts of the different segments Figure , and theThe 3' and 5' end sequences of the NCRs of RNA segments of ISAV were confirmed from RNA extracted from purified virus, i.e., vRNA, using 3' and 5' RACE, respectively, and appropriate primers Tables  and 7. FOrthomyxoviridae are GCU-3' (except in ISAV segment 5 with ACU-3'), and the length of the non-coding region in orthomyxoviruses other than ISAV is 20-23 nucleotides long [The length of the 3' NCR in the different genome segments was variable, ranging from seven nucleotides in segment 6 to 48 nucleotides in segment 3 Figure . The firdes long -54. TablOrthomyxoviridae [The length of the 5' NCR in all eight ISAV segments was significantly longer than the corresponding 3' NCR Figure . It was oviridae ,15. In bThe sequences of 3' and 5' terminal sequences of genomic segments of orthomyxovirus are partially complementary, which results in the formation of double helical structures known as panhandle structures . The preThe present study focussed on the quasispecies distribution found in the NCRs of mRNA/cRNA of each genome segment of ISAV. Comparison of 3' UTR sequences of mRNA transcripts from the different segments revealed heterogeneity among the five different clones of the same segment for all RNA segments. The 5' and 3' NCRs of the cRNA from the different segments were also variable; the NCRs of segment 5 cRNA were the least variable with all five clones showing identical sequence, whereas the 3' NCR of segment 3 cRNA had the highest HI (0.8) of all NCRs of ISAV cRNA, with all five clones having different sequences Table  and 5. MThe biological significance of quasispecies in the terminal NCR sequences of ISAV is not known at this time. We speculate that the quasispecies detected may play an important role in ISAV replication. For replication in influenza virus, it is known that viral RNA dependent RNA polymerase initiates the RNA synthesis on viral RNA by binding to the panhandle structure formed as a result of partial complementarity of 3' and 5' non-coding sequences of viral RNA . TherefoOrthomyxoviridae are GCU-3' (except in ISAV segment 5 with ACU-3').We report for the first time a comprehensive unambiguous analysis of NCR sequences of all eight RNA genome segments from two strains of ISAV belonging to the two ISAV genotypes. The experimental design used Figure, wherebyT/ATTTTTACT-3' (in the message sense 5'-3') in all segments except for segment 3 at position 4 with a T→A mutation, segments 3, 4, 5, and 7 at position 9 with T→A mutation, segment 8 at position 10 with T→A mutation, and segment 7 at position 11 with a C→G mutation. A BLAST search [Orthomyxoviridae [However, a BLAST search  of the GT search  of the GT search  revealedT search ,28 and 7T search ,33. ThisT search . The seqT search  ranged foviridae . Thus thoviridae ,59,60. BIt is interesting that in the quasispecies study, all five clones of the 3' NCR of segment 5 cRNA of ADL-ISAV-07 had identical sequence (HI = 0.0), and yet it was the most divergent between the two ISAV genotypes at 77.2% sequence identity. This is even more divergent than at the amino acid level . ClearlyT/ATTTTTACT-3' (in the message sense 5'-3') in all segments of both ISAV genotypes.In conclusion, using sequence analysis of multiple clones derived from one RT-PCR product for each mRNA/cRNA transcript end for the 8 RNA genome segments, we present evidence of intra-segment ISAV quasispecies, with some RNA segments being more prone to genetic changes in their transcripts. The 3' NCR sequence of segment 5 was the least divergent within the viral population but was the most divergent between the two ISAV genotypes. Conversely, the viral population of 3' NCR sequence of segment 3 cRNA transcripts was the most heterogenous but the consensus sequence was identical between the two ISAV genotypes. Moreover we report for the first time the comprehensive unambiguous identification of the 5' and 3' terminal sequences of the 8 RNA genome segments from two strains of ISAV representing the two genotypes of ISAV. Because most ISAV sequences are of cDNA to mRNA, they do not contain the 3' end sequences, which are removed during polyadenylation of the mRNA transcripts. We report for the first time the ISAV consensus sequence CAISAV strains NBISA01 of North American genotype, and ADL-PM 3205 ISAV-07 of the European genotype were used in this study. NBISA01 was propagated in CHSE-214 cell line whereas ADL-PM 3205 ISAV-07 was propagated in the macrophage/dendritic like cell line (TO cell line), and the lysates were harvested as described ,62.Total RNA was extracted from ISAV-infected TO cells, and vRNA was extracted from purified virus by treating the samples with 1.2 ml of TRIzol reagent (Invitrogen) for 10 minutes, and then adding 300 ul of chloroform followed by centrifugation at 12,000 rpm for 15 minutes. The aqueous phase was loaded on RNeasy Mini Columns and subsequent steps were carried out as per the protocols of RNeasy Mini Kit (Qiagen). The extracted RNA was subsequently treated with DNAse I to remove the contaminating DNA using the RNase-free DNAse I (Qiagen). The purity of RNA was examined using A260/280 ratio based on spectrophotometer readings.Attempts were made to use the rapid amplification of cDNA ends (RACE) method to determine the 5' and 3' NCRs of the ISAV genome.The 5' RACE was performed using a commercially available kit from Invitrogen. In this method, first strand cDNA was synthesized from RNA with M-MLV enzyme (Invitrogen) using the ISAV segment specific primers (ssp1) Table . The synThe 3' RACE was performed using a commercially available kit from Invitrogen. In this method, the viral RNA was first polyadenylated by treating the RNA with poly-A-polymerase (Ambion) at 37°C for an hour. The viral RNA was purified using NucAway spin column (Ambion) and used as a template for 3' RACE. The polyadenylated RNA was converted to cDNA using an oligo-dT primer and reverse transcriptase enzyme. The cDNA was then amplified using UAP and either ISAV segment specific primers (ssp3) for total RNA Table  or ISAV 2, 200 uM dNTPs, 2.5 U Expand High Fidelity DNA polymerase (Roche) and forward and reverse ISAV segment specific primers (Tables The 3' and 5' ends of ISAV RNA were ligated using T4 RNA Ligase (Invitrogen). A duplicate sample of viral RNA was first treated with tobacco acid pyrophosphatase (TAP) (Epicenter) at 37°C for 1 h. It was then purified using NucAway spin column (Ambion) prior to the ligation reaction. RNA (14 ul) was ligated in a total volume of 20 ul in 1 × X T4 RNA Ligase buffer, 40 units of RNase out (Invitrogen) and 40 units of T4 RNA Ligase. The mixture was incubated at 37°C, followed by heat inactivation at 65°C for 15 min. The cDNA was synthesized from circularized RNA using the Superscript III First Strand Synthesis System for RT-PCR (Invitrogen). Subsequently the PCR was carried out in a final volume of 50 ul containing 1X Expand HiFi reaction buffer, 1.5 mM MgCls Tables , each atEcoRI and plasmid DNA sequencing was performed on EcoRI positive clones by ACGT Corporation (Ontario). The cDNA sequences were deposited in the GenBank Database [The purified PCR products were cloned in the plasmid pCRII-TOPO using the TOPO TA cloning kit (Invitrogen). The clones were screened by restriction enzyme analysis of plasmid DNA with Database .Sequences were analysed using the Sequence Manipulation Suite program  and the KS, NS, and AR are employees of Novartis Animal Health Canada Inc. This does not alter the authors' adherence to all the BioMed Central policies on sharing data and materials.VK carried out the molecular genetic studies, performed the sequence alignment and drafted the manuscript. MK participated in the original design of the study, prepared the purified virus, and drafting of the manuscript. KS, NS, and AR participated in the original design of the study, and drafting of the manuscript. FK conceived of the study, and participated in its design and coordination, and drafted the manuscript. All authors read and approved the final manuscript.