Our work defines previously unappreciated viral regulatory elements and provides the first in-depth view of the intracellular genome dynamics of this emerging human pathogen.Approximately 20 million hepatitis E virus (HEV) infections occur annually in both developing and industrialized countries. Most infections are self-limiting, but they can lead to chronic infections and cirrhosis in immunocompromised patients, and death in pregnant women. The mechanisms of HEV replication remain incompletely understood due to scarcity of adequate experimental platforms. HEV undergoes asymmetric genome replication, but it produces an additional subgenomic (SG) RNA encoding the viral capsid and a viroporin in partially overlapping open reading frames. Using a novel transcomplementation system, we mapped the intragenomic subgenomic promoter regulating SG RNA synthesis. This  HEV is an emerging pathogen causing severe liver disease. The genetic information of HEV is encoded in RNA. The genomic RNA is initially copied into a complementary, antigenomic RNA that is a template for synthesis of more genomic RNA and for so-called subgenomic RNA. In this study, we identified the precise region within the HEV genome at which the synthesis of the subgenomic RNA is initiated. The nucleotides within this region are conserved across genetically distinct variants of HEV, highlighting the general importance of this segment for the virus. To identify this regulatory element, we developed a new experimental system that is a powerful tool with broad utility to mechanistically dissect many other poorly understood functional elements of HEV. In 1983, the first experimental evidence was provided for an additional waterborne hepatitis agent distinct from hepatitis A virus (HAV) that was eventually named hepatitis E virus (HEV) . HEV preHepeviridae family (HEV is a positive-sense (+), single-stranded, quasienveloped icosahedral RNA virus belonging to the e family , 5. The e family . ORF1 coe family . ORF2 ise family .cis-acting element regulating subgenomic (SG) RNA synthesis is and what the kinetics and dynamics of the different viral RNAs  are. These are critical questions, and answering them will provide important insights into the mechanism of HEV genome replication.Following viral entry into host cells and uncoating of the viral genome, the (+) RNA is translated by host ribosomes into a polyprotein containing the nonstructural enzymes of the virus, including the RdRp. This HEV RdRp then transcribes the (+) RNA into a complementary negative-sense (−) RNA. Consistent with presumed asymmetric genome replication, the full-length (FL) (−) strand genome serves as a template for more (+) RNA and for synthesis of the SG RNA(s) encoding ORFs 2 and 3. Transcription of the (+) and (−) RNA is initiated at secondary RNA structures at the respective 5′ and 3′ UTRs. Nucleotides on the stem-loop RNA structure in the junction region (JR) between the stop codon of ORF1 and the start codons of ORFs 2 and 3 have been implicated in viral genome replication . Howevercis-acting element functioning as the promoter to direct HEV subgenomic RNA synthesis. We devised a novel experimental system enabling us to interrogate the functional relevance of specific nucleotides within the FL genome by complementing the ORF1 function in trans. Using this novel transcomplementation system, we precisely mapped the region within the viral (−) RNA that is critical for transcription of the HEV subgenome. This subgenomic promoter (SgP) spans a 44-nucleotide region from the 3′ end of the ORF1 to the transcriptional start site (TSS) of ORF3   that is critical for SG RNA synthesis off the FL (−) strand, we devised an experimental system in which HEV RNA transcription is uncoupled from protein translation. Following our previously established successful transcomplementation approach for studying HEV ORF3’s viroporin function , we expr6 genome  in whiche (Gluc) , termed e (Gluc) . Gluc ace (Gluc) . Importaee trans . Consistee trans , deletioUncoupling of HEV RNA transcription from protein translation opened opportunities to map the SgP producing the SG RNA encoding HEV ORF2 and ORF3. Sequences identical or very similar to those at the respective 5′ and 3′ UTRs forming secondary RNA structures that regulate transcription of the (+) and (−) RNA are not present in other parts of the genome. Thus, we followed an unbiased approach and first screened a larger region upstream of the subgenomic RNA transcriptional start site , 12 for To more accurately map the relevant sequence, we created additional mutant rHEVΔORF2/3[Gluc]-Pol- genomes lacking 10-nt segments between positions 5225 to 5339 nt . Similarcis-acting element within the viral genome critical for regulating transcription of subgenomic HEV RNA.Conceivably, deleting these—albeit—small regions within the HEV genome could possibly affect the efficiency of HEV (−) strand RNA synthesis and/or stability of the negative strand, thereby indirectly affecting SG RNA synthesis. To rule out this possibility, we devised a sensitive negative-strand RNA quantitative reverse transcription-PCR (RT-PCR) detection assay. Using this strand-specific quantitative RT-PCR assay , we demo10.1128/mBio.00769-18.1FIG S1 in vitro-transcribed RNA using the strand-specific RT-qPCR. cDNAs synthesized by reverse transcription of negative-strand viral RNA genome in the presence or absence of a fixed amount of in vitro transcript positive- or negative-strand viral RNA genome using tagged RT primer were quantified using qPCR. The experiment was performed in triplicate, and mean threshold cycle (CT) values with standard deviation were plotted against RNA absolute copy number. The correlation coefficients of the qPCRs are presented below each curve. Download FIG S1, TIF file, 3.3 MB.Strand-specific RT-qPCR assay to quantify the HEV negative-strand RNA genome. (a) Schematic illustration of the positions of the primers in the RT-qPCR assay. For the RT primer  Schematic representation of the ORF1 transcomplementation system. (b) Representative flow cytometry plots demonstrating efficient ORF1Flag transduction. HepG2C3A cells were transduced with pLVX-ORF1Flag-IRES-zsGreen (WT or GAD mutant) or not transduced. Flow cytometric analysis was performed 3 days following transduction to quantify the frequencies of ORF1-expressing cells. (c) The ORF1 Flag (WT or GAD mutant)-transduced HepG2C3A cells were subjected to immunoblotting to analyze the expression of ORF1Flag with Flag antibody. (d) Replication kinetics of HEV RNA in ORF1 transcomplemented HepG2C3A cells. Cell culture supernatants from naive HepG2C3A cells or HepG2C3A cells transduced with epitope-tagged HEV ORF1 or its GAD mutant were collected at the indicated time points posttransfection with rHEVΔORF2/3[Gluc] Pol- RNA or its mutants RNA, and Gaussia luciferase activity were quantified. Data represent the means plus SD (n = 3). ***, P < 0.001 by two-tailed Student’s t test. Download FIG S2, TIF file, 5.6 MB.HEV Flag-tagged ORF1 is able to function in Copyright © 2018 Ding et al.2018Ding et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the M73218  reference genome) showed that this cis-acting element is highly conserved across genetically diverse HEV genotypes 1 to 8 (mut1 (magenta line) compared to the parental sequence (Mut2) or 4 nucleotides (rHEV Gluc SgPMut3). Similar to the rHEV Gluc SgPMut1 harboring 14 synonymous mutations, Gluc activity was reduced 500- to 1,000-fold for both the sgPMut2  reduced luciferase activity to background levels.To provide direct experimental evidence that mutations in the SgP also affect SG RNA transcription of other, genetically diverse HEV genotypes, we expanded our analysis to SAR55 (genotype [gt] 1) , 15, pSHompetent . Of noteompetent  . *, P < 0.05; ***, P < 0.001 by two-tail Student’s t test. Download FIG S3, TIF file, 10.5 MB.Generation and characterization of HEV reporter genomes. Schematic diagrams of the rHEVΔORF2/3[Gluc] genomes. S17 sequences were inserted into the X domain of ORF1 as described. GAD mutant was generated as the negative control. rHEVΔORF2/3[Gluc] WT, S17 inserted or GAD mutant RNA were delivered into Huh 7.5 cells. Cell culture medium was collected after 4 days transfection, and Copyright © 2018 Ding et al.2018Ding et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the cis-acting element, then mutations in this region abrogating this function would severely impair particle production and release, as the SG RNA is the translational template for ORF2 capsid and the ORF3 viroporin. To test this hypothesis directly, we introduced the same synonymous mutations in the FL Kernow C1 p6 (genotype 3) and FL TW6196E (genotype 4) genomes while retaining the ORF1 protein sequence  analysis proved that Huh7.5 cells exposed to the SN from FL Kernow C1 p6 wt, but not from the SgPmut (or Pol−) mutant, became infected  of the HEV genome harbor a region that is critical for subgenomic RNA synthesis and ultimately production and release of infectious virions.If HEV SG RNA synthesis were truly dependent on this sequence . In vitrinfected . These dinfected . These dM73218  reference genome) controls synthesis of the SG RNA across multiple different HEV genotypes. Prior seminal work on, e.g., alphaviruses, showed that in addition to a region(s) 5′ of the transcription start site (TSS), residues 3′ of the TSS also serve essential functions for producing Sindbis virus (SINV) subgenomic RNA  encompass the promoter for subgenomic RNA synthesis.To narrow down further which of the 9 nt are of particular importance, we created three additional rHEVΔORF2/3[Gluc]-Pol- harboring deletions of nt 1 to 3, nt 4 to 6, or nt 7 to 9 and tested them in our transcomplementation assay. Irrespective of the triplet mutation, luminescent activity was greatly reduced  binds to the 3′ untranslated region (3′ UTR) of HEV and transcribes complementary, full-length (FL) negative-sense (−) viral RNA  which se19–In future studies, it will be of great interest to determine the dynamics of the HEV (+) and (−) strand and subgenomic RNAs both longitudinally and across different tissue and cell types. Conceivably, albeit unlikely given the high degree of sequence conservation of the SgP region, HEV genotypes primarily infecting zoonotic hosts  may have different RNA kinetics, which could be tested as we gain access to the appropriate materials and cells from the primary host species .trans, we were able to interrogate the impact of specific nucleotide deletions within the RdRp region of ORF1. Thus, a similar analysis could not have been performed in the context of the FL viral genome. Our system also overcomes several notable challenges to studying HEV, such as its limited replication efficiency in vitro. By delivering ORF1 in trans with a genome carrying a highly sensitive Gluc reporter, we created a simple readout for evaluating the effect of introducing mutations in the SgP region. We anticipate that this new transcomplementation system will be widely useful to study other aspects of ORF1 function, including but not limited to its effect on host specificity and its ability to interact with different viral elements. ORF1 is not only the largest but arguably also the most complex gene product encoded by HEV. Numerous distinct units have been predicted within ORF1 , HepG2C3A cells  and Huh7.5 cells  were maintained in Dulbecco’s modified Eagle medium (DMEM)  supplemented with 10% (vol/vol) fetal bovine serum (FBS), 50 IU/ml penicillin and streptomycin, in a humidified 5% (vol/vol) COM73218 , M74506 , JQ679013.1 , AB197673 (4a), AB573435 (5a), AB856243 , KJ496144 , and KX387866 . M73218  was used as the reference strain for numbering. A ClustalW multiple-sequence alignment was performed on regions containing the putative subgenomic promoter (SgP), using MacVector software version 15.1.5.The following hepatitis E virus (HEV) strains were used for sequence alignment: GenBank identifiers [IDs] or accession numbers JQ679013), the Kernow C1/p6 ORF1 cDNA was amplified by PCR from a plasmid encoding the full-length (FL) infectious HEV clone Kernow C1/p6  and then cloned into pLVX-IRES-zsGreen1 vector using an In-Fusion HD cloning kit . The GAD mutant of ORF1 inactivating the polymerase was generated by QuikChange (Stratagene) site-directed mutagenesis. The pKernow-C1 p6/Gluc  with HEV pKernow-C1 p6-Gluc as the template by PCR, and PU-O-4339 and PU-O-4340 were used as primers to produce DNA fragment 3 with pSK-SAR55 as the template by PCR assay. These three DNA fragments were assembled into pSK-SAR55 (SfiI- and EcoRI-digested) vector. To generate the pSK-SAR55(S17)-Gluc reporter construct, PU-O-4871 and PU-O-4338 were used as primers to produce DNA fragment 1 with pSK-SAR55(S17) as the template by PCR, and PU-O-2783 and PU-O-4340 were used as primers to produce DNA fragment 2 (containing the Gluc reporter gene) with pSK-SAR55-Gluc as the template by PCR. These two DNA fragments were assembled into pSK-SAR55(S17) (EcoRI-digested) vector. To make the pSK-SAR55-Gluc GAD mutant, QuikChange (Stratagene) site-directed mutagenesis was performed using pSK-SAR55-Gluc as the template and PU-O-4635 and PU-O-4636 as the primers.To generate the pKernow-C1 p6-Gluc reporter construct without the S17 insertion, PU-O-4031 and PU-O-4261 were used as the primers to produce DNA fragment 1 using pKernow-C1 p6-Gluc as the template by PCR, and PU-O-4260 and PU-O-4322 were used as the primers to produce DNA fragment 2 using pKernow-C1 p6-Gluc as the template by PCR assay. Both DNA fragments were assembled into pKernow-C1 p6-Gluc vector . To make the pKernow-C1 p6-Gluc GAD construct, QuikChange (Stratagene) site-directed mutagenesis was performed using pKernow-C1 p6-Gluc as the template and PU-O-2711 and PU-O-2712 as the primers.To generate the pGEM-9Zf-pSHEV3-Gluc reporter construct, PU-O-4327 and PU-O-4328 were used as primers to produce DNA fragment 1 using pGEM-9Zf-pSHEV3 as the template by PCR assay, and PU-O-2783 and PU-O-4329 were used as the primers to produce DNA fragment 2 (containing the Gluc gene) using Kernow-C1 p6/Gluc as the template by PCR assay. These DNA fragments assembled into pGEM-9Zf-pSHEV3 (AflII- and PmlI-digested) vector. To generate the pGEM-9Zf-pSHEV3(S17)-Gluc reporter construct, PU-O-4844 and PU-O-4845 were used as primers to produce DNA fragment 1 with pGEM-9Zf-pSHEV3-Gluc as the template by PCR, PU-O-4846 and PU-O-4847 were used as primers to produce DNA fragment 2 (containing the Gluc reporter gene) with HEV pKernow-C1 p6/Gluc as the template by PCR, and PU-O-4848 and PU-O-4849 were used as primers to produce DNA fragment 3 with pGEM-9Zf-pSHEV3-Gluc as the template by PCR assay. These three DNA fragments were assembled into pGEM-9Zf-pSHEV3-Gluc (StuI-digested) vector. To make the pGEM-9Zf-pSHEV3-Gluc GAD construct, QuikChange (Stratagene) site-directed mutagenesis was performed using pGEM-9Zf-pSHEV3-Gluc as the template and PU-O-4376 and PU-O-4377 as the primers.To generate the pGEM-7Zf(-)-TW6196E-Gluc reporter construct, PU-O-4330 and PU-O-4331 were used as primers to produce DNA fragment 1 using pGEM-7Zf(-)-TW6196E as the template by PCR assay, PU-O-2783 and PU-O-3934 were used as the primers to produce DNA fragment 2 (containing the Gluc gene) using Kernow-C1 p6/Gluc as the template by PCR assay, and PU-O-4332 and PU-O-4333 were uses as the primers to produce DNA fragment 3 using pGEM-7Zf(-)-TW6196E as the template by PCR assay. These DNA fragments assembled into the pGEM-7Zf(-)-TW6196E (Sbf-I and EcoRI-digested) vector. To generate the pGEM-7Zf(-)-TW6196E(S17)-Gluc reporter construct, PU-O-4850 and PU-O-4851 were used as primers to produce DNA fragment 1 using pGEM-7Zf(-)-TW6196E-Gluc as the template by PCR assay, and DNA fragment 2 containing S17 element and partial pGEM-7Zf(-)-TW6196E sequences were synthesized. These DNA fragments were assembled in the pGEM-7Zf(-)-TW6196E-Gluc (ApaI digested) vector. To generate the pGEM-7Zf(-)-TW6196E-Gluc GAD construct, QuikChange (Stratagene) site-directed mutagenesis was performed using pGEM-7Zf(-)-TW6196E-Gluc as the template and PU-O-4631 and PU-O-4632 as the primers.All DNA fragments were cloned into the respective vectors using an In-Fusion HD cloning kit . All constructs or primers used to construct the HEV reporter genome in 5 cells/well) were seeded in collagen-coated six-well tissue culture plates and infected the following day with lentiviruses. To determine the transduction efficiency, cells were trypsinized and processed for flow cytometric analysis 3 days following transduction.Vesicular stomatitis virus G protein (VSV-G) pseudotyped lentiviruses were produced by transient cotransfection of the third-generation packaging plasmids pMD2G  and psPAX2  and the transfer vector with XtremeGENE HP DNA transfection reagent  into HEK293T cells. The medium was changed 6 h posttransfection. Supernatants were collected at 48 and 72 h after transfection, pooled, passed through a 0.45-µm filter, aliquoted, and frozen at −80°C. For lentiviral transduction, HepG2C3A cells . Kernow C1/p6 ORF1 or ORF1 GAD were transduced into target cells by bicistronic lentiviruses expressing zsGreen. After 3 days of transduction, cells were fixed in 4% (wt/vol) paraformaldehyde (PFA) in phosphate-buffered saline (PBS) for 15 min, following by two washes with PBS. The efficiencies of transduction of transgenes were determined by simultaneous expression of zsGreen. Quantification of the HEV-infected cells was performed using ORF2-specific convalescent-phase serum collected from an HEV-infected chimpanzee . Briefly, supernatant from the HEV RNA-transfected cells was used to inoculate naive Huh 7.5 cells for 12 h. The cells were washed with PBS three times and subsequently cultured in DMEM with 10% (vol/vol) FBS. After 3 days of infection, the cells were trypsinized, and single-cell suspensions were fixed with 4% (wt/vol) PFA and permeabilized in PBS plus 0.01% Triton X-100, following two washes with PBS. The cells were stained with anti-ORF2 serum CH1313 (1:100) for 1 h at room temperature, followed by two washes with PBS. Cells were subsequently incubated with goat anti-human IgG secondary antibody conjugated with Alexa Fluor 488 (Life Technologies) at a 1:1,000 dilution for 1 h at room temperature. All samples were analyzed on a BD LSRII flow cytometer using FlowJo software.in vitro from linearized plasmid using HiScribe T7 antireverse cap analog (ARCA) mRNA kit  according to the manufacturer’s instructions. The in vitro transcription (IVT) reaction mixture of 20 µl was assembled by adding DNA template (1 µg), 10 µl of 2×ARCA/nucleotide triphosphate (NTP) mix and 2 µl of T7 RNA polymerase Mix. The reaction mixture was incubated at 37°C for 2 h, and 1 µl of DNase was added to the IVT reaction mixture and incubated for 15 min at 37°C to remove the template DNA. Then, the viral RNA was purified by LiCl precipitation. Viral RNA was transfected into HepG2C3A cells or Huh7.5 cells using TransIT-mRNA transfection reagent  according to the instructions.HEV Kernow-C1 p6, HEV Kernow-C1 p6-Gluc, and the truncated mutant plasmids were linearized by MluI. pSAR55-Gluc was linearized by BglII, pGEM-9Zf-pSHEV3-Gluc was linearized by XbaI, and pGEM-7Zf(-)-TW6196E and pGEM-7Zf(-)-TW6196E-Gluc were linearized by SpeI. Viral capped RNAs were transcribed Gaussia luciferase activity was determined using Luc-Pair Renilla luciferase HS assay kit . Specifically, 10 µl of harvested cell culture medium was added per well of a 96-well solid white, flat-bottom polystyrene microplate , followed by the addition of Renilla luciferase assay substrate and the detection of luminescence was performed using a Berthold luminometer.in vitro-transcribed viral capped RNAs (2 µg) were transfected into Huh7.5 cells (2 × 105 cells) in the six-well tissue culture plates using TransIT-mRNA transfection reagent . Medium was removed from transfected cells after 5 days transfection, and cells were trypsinized and washed with PBS once. Cell pellets were extracted by adding 1 ml double-distilled (DDI) water per well and vortexing vigorously until the cell pellets were completely suspended. The sample was put on ice for 30 min, vortexing intermittently every 10 min. The samples were centrifuged at 13,000 × g for 10 min to remove cellular debris. Supernatant (0.9 ml) was transferred to a new tube, and 0.1 ml of 10× PBS was added. The supernatant was stocked at −80°C. As for the infection assay, the Huh7.5 cells were seeded into a 24-well tissue culture plate 24 h prior to infection, and cells were infected by HEV the following day, and medium was changed 12 h after the infection. After 3 days, cells were collected for further analysis.The in vitro transcription assay to produce the negative-strand viral RNA. The in vitro transcription assay was performed using HiScribe T7 ARCA mRNA kit (New England Biolabs) as described above. The RNA templates were purified and stocked at −80°C.To prepare the RNA standard used to quantify the Kernow C1/p6 Gluc negative-strand RNA abundance, DNA fragment spanning from 4751 nt to 6924 nt of Kernow C1/p6 Gluc was amplified by PCR and then subcloned into the pCR4BluntTOPO vector (Thermo Fisher Scientific) under the control of the T7 promoter. The correct insert direction clones were identified by DNA sequencing as the templates for an Series-diluted predetermined copies of standard RNA or 100 ng of total RNA from each sample were used for reverse transcription using SuperScript III reverse transcriptase . The reverse transcription primer (PU-O-4508) contained a nonviral tag sequence attached at the 5′ end of the strand-specific viral sequence that was used to prime reverse transcription . The RT in vitro-transcribed standard RNA. The viral genome copy numbers were determined by interpolation of the standard curve for the respective strand of RNA.Real-time qPCRs was prepared using SYBR green PCR master mix . Briefly, 2 µl of diluted cDNA was mixed with 2×SYBR green PCR master mix and the primers (PU-O-4509 and PU-O-4125)  at a fint test or one-way analysis of variance (ANOVA) with Tukey’s honestly significant difference (HSD) test was used to test for statistical significance of the differences between the different group parameters. P values of less than 0.05 were considered statistically significant.Student’s


Paris polyphylla Smith var. yunnanensis (Franch.) Hand. -Mazz. and Paris polyphylla Smith var. chinensis (Franch.) Hara are the two main sources of Chonglou under the monograph of Rhizoma Paridis in Chinese Pharmacopoeia. In the local marketplace, however, this medicine is prone to be accidentally contaminated, deliberately substituted or admixed with other species that are similar to Rhizoma Paridis in shape and color. Consequently, these adulterations might compromise quality control and result in considerable health concerns for consumers. This study aims to develop a rapid and sensitive method for accurate identification of Rhizoma Paridis and its common adulterants.Rhizoma Paridis (Chonglou) is a commonly used and precious traditional Chinese medicine. P. polyphylla var. chinensis.DNA barcoding coupled with high resolution melting analysis was applied in this research to distinguish Rhizoma Paridis from its adulteration. The internal transcribed spacer 2 (ITS2) barcode was selected for HRM analysis to produce standard melting profile of the selected species. DNA of the tested herbal medicines was isolated and their melting profiles were generated and compared with the standard melting profile of The results indicate that the ITS2 molecular regions coupled with HRM analysis can effectively differentiate nine herbal species, including two authentic origins of Chonglou and their seven common adulterants. Ten herbal medicines labeled “Chonglou” obtained from a local market were collected and identified with our methods, and their sequence information was analyzed to validate the accuracy of HRM analysis.DNA barcoding coupled with HRM analysis is a accurate, reliable, rapid, cost-effective and robust tool, which could contribute to the quality control of Rhizoma Paridis in the supply chain of the natural health product industry (NHP).The online version of this article (10.1186/s13020-018-0162-4) contains supplementary material, which is available to authorized users. Paris polyphylla Smith var. yunnanensis (Franch.) Hand. -Mazz. and P. polyphylla Smith var. chinensis (Franch.) Hara, according to the Chinese Pharmacopoeia . Eight species can be clearly distinguished by the NJ tree , P. thibetica, P. tengchongensis, P. forrestii, P. mairei, T. tschonoskii and P. paleaceum. The genetic relationships reflected by the NJ tree were in good agreement with the Bar-HRM results. The sequence of each amplicon and the alignment among eight species are shown in Fig. To validate the results of HRM analysis, amplicon sequences of all samples from the nine reference plant species were sequenced. Twenty-seven ITS2 sequences from eight species were successfully obtained, except for Tm values and GCPs of ten samples obtained from the HRM analysis are shown in Tables Tms of five Chonglou samples  ranged from 87.29 to 87.33 °C, which agreed with the Tm of P. polyphylla var. yunnanensis, and the curves overlapped completely. These results demonstrated that all these five samples were P. polyphylla var. yunnanensis. Among them, three samples  were identified as P. tengchongensis. Com-7 sharing a similar plot with T. tschonoskii. Com-9 was identified as P. mairei. These results indicate that Bar-HRM analysis can be effectively used to differentiate Chonglou from its adulterants.As mentioned previously, Chonglou sold in the local market places can be confused with certain other species that are similar in shape and color with the authentic Chonglou. To evaluate the accuracy of Bar-HRM method and to assure the quality of Chonglou in local market, the proposed methodology was further applied to authenticate ten batches of samples labelled as “Chonglou”. The HRM curves of nine reference plant species and ten samples are shown in Fig. Species identification is critical to ensuring the quality of herbal medicine. HRM analysis is a method that measures the dissociation rates of double-stranded DNA into single-stranded DNA in increasing temperatures . Minute Paris species [Paris species [At present, several methods, such as high performance liquid chromatography (HPLC) and near infrared spectroscopy (NIR), have been introduced to discriminate the controversial  species , 28. How species . Among t species . HoweverParis species and their seven adulterants. The results indicated that Bar-HRM is a sensitive, fast, cheap, and reliable method for identifying and tracking herbal medicines. To validate the accuracy of polymorphisms detected by HRM analysis, the sequences of the reference plant specie were analyzed. Each PCR product was amplified and both strands were sequenced in opposite directions. Although the PCR products of Tupistra spp. were clearly visible as a single band on agarose gel, the sequence information of Tupistra spp. was not obtained, and there are no documented reports on its ITS2 sequences information in the GeneBank. The multi-copy of ITS2 region may lead to the failure of sequence. However, the HRM melting curves could be used to distinguish Chonglou from its adulterants clearly, included Tupistra spp. Considering these features, Bar-HRM can be a useful tool for quality control of herbal medicines in the supply chain of the natural health product industry (NHP) and medicine supervisory institutions. In conclusion, this study substantially contributes to the protection of public health worldwide.In this study, we have successfully applied Bar-HRM analysis for the rapid detection and identification of two major P. polyphylla var. yunnanensis and P. polyphylla var. chinensis, from their adulterate species. Species-specific PCR amplification of the ITS2 region was performed to validate the results of HRM analysis. The difference between Chonglou species and their adulterants can be observed from their sequencing information. The proposed method has been proved to be accurate, reliable, rapid, cost-effective and robust for identifying Chonglou from its adulterants. Bar-HRM analysis is also feasible for high-throughput assays, and could be well applied to other herbal species in the future, ensuring the perceived value and quality of herbal medicines.The Bar-HRM analysis has been used in this study to differentiate Chonglou, namely Additional file 1. Minimum Standards of Reporting Checklist.


Aurora-A mRNA, either in a cap-dependent or -independent manner, by interacting with the 5′-UTR of Aurora-A mRNA through its RNA-binding domains (RBDs) 2 and 3. By ribosomal profiling assay further confirmed the translational regulation of Aurora-A mRNA by hnRNP Q1. Overexpression of hnRNP Q1 promotes cell proliferation and tumor growth. HnRNP Q1/ΔRBD23-truncated mutant, which loses the binding ability and translational regulation of Aurora-A mRNA, has no effect on promoting tumor growth. The expression level of hnRNP Q1 is positively correlated with Aurora-A in colorectal cancer. Taken together, our data indicate that hnRNP Q1 is a novel trans-acting factor that binds to Aurora-A mRNA 5′-UTRs and regulates its translation, which increases cell proliferation and contributes to tumorigenesis in colorectal cancer.By using RNA-immunoprecipitation assay following next-generation sequencing, a group of cell cycle-related genes targeted by hnRNP Q1 were identified, including Aurora-A kinase. Overexpressed hnRNP Q1 can upregulate Aurora-A protein, but not alter the mRNA level, through enhancing the translational efficiency of  HnRNP Q members consist of one acidic domain (AcD) at the N-terminus that is involved in protein–protein interaction, three RNA-binding domains (RBDs) in the central region and one RGG box at the C-terminus for binding to RNA. HnRNP Q proteins have multifunction in regulating mRNAs, including pre-mRNA splicing,5 mRNA editing,7 transport,9 turnover11 and primarily regulating translation.14 Among the hnRNP Q proteins, hnRNP Q1  is the smallest isoform, ubiquitous and the most abundant member of the family. HnRNP Q1 lacks the second nuclear localization signals (NLS) in the C-terminus and has a subcellular localization in both the cytoplasm and nucleus, differing from the other hnRNP Q isoforms which are limited to the nucleus.5 The cytoplasmic localization of hnRNP Q1 may respond to most of the mRNA metabolic processes occurring in the cytoplasm, including mRNA transport, translation and turnover, all of which can directly affect the protein production of target mRNAs.Heterogeneous nuclear ribonucleoproteins (hnRNPs) are a large group of RNA-binding proteins that associate with the heterogeneous nuclear RNAs (hnRNAs) transcribed by RNA polymerase II in eukaryotic cells. All members of the hnRNP family share a similar protein structure, consisting of at least one RNA-binding domain  and combine with other auxiliary domains such as the RGG box or the acidic domain responsible for protein–protein interactions or additional RNA-binding abilities.15 However, some reports indicate that hnRNP Q can target the 3′-UTR of mRNAs and then disrupt poly(A)-binding protein (PABP)-mediated circular mRNA formation to repress cap-dependent translation.17 Therefore, the role of hnRNP Q in regulating translation might depend on the binding sites of mRNAs. A previous report indicated that hnRNP Q is involved in the maintenance of cell proliferation in colon cancer cells by associating with galectin-3, but the underlying mechanism is still unclear.18 Furthermore, the specific target genes regulated by hnRNP Q have not been completely identified.The internal ribosome entry site (IRES) is a specific secondary structure located at the 5′-UTR of mRNA. The IRES can recruit a ribosome to initiate translation without the formation of 5′-cap recognized complex. During mitosis, cap-dependent translation is suppressed, and IRES-mediated translation is the alternative way for protein synthesis. According to the literatures, hnRNP Q can act as a positive regulator of the IRES.Aurora-A mRNA can be upregulated in colorectal cancer by membrane receptor-mediated downstream signaling, and the 5′-UTR of Aurora-A mRNA is critical for translational regulation.19 In the present study, we found that hnRNP Q1 can promote cell proliferation and translationally regulates a group of cell cycle-related genes including Aurora-A. The expression of hnRNP Q1 is positively correlated with Aurora-A in human colorectal cancer tissues. Our results suggest that hnRNP Q1 is a novel trans-acting factor that binds to the Aurora-A 5′-UTR and regulates translation, both in cap-dependent and IRES-dependent manners, which may increase cell proliferation and contribute to the tumorigenesis of colorectal cancer.Our previous report showed that the translation of To investigate the role of hnRNP Q1 in tumorigenesis, the expression of hnRNP Q1 in colorectal cancer cell lines was first examined. The results indicated that hnRNP Q1 is overexpressed in colorectal cancer cell lines than the normal colon cell line CRL1790 . To furt10 Interestingly, a group of cell cycle- and mitosis-related genes are the potential targets of hnRNP Q1 (Aurora kinase A (AURKA) for the following study because of its roles in mitotic entry and tumorigenic capacity.20To further identify the hnRNP Q1-associated mRNAs in cancer cells, an RNA-immunoprecipitation (IP) assay followed by next-generation sequencing (NGS) was performed in GFP-hnRNP Q1-overexpressed cells. The subcellular localization of hnRNP Q1 was first characterized, and the results indicated that only hnRNP Q1 existed in both the nucleus and the cytoplasm, in contrast to the other hnRNP Q variants, hnRNP Q2 and Q3 . SupplemhnRNP Q1 . This reAurora-A mRNA isoforms that result from alternative splicing of the 5′-UTR  showed a stronger binding affinity with hnRNP Q1. The interaction between endogenous hnRNP Q1 and Aurora-A mRNA 5′-UTRs was confirmed by RNA-IP assay  has an impaired interaction ability with Aurora-A mRNA  binding analysis was performed to identify the NA probe . The rest manner . In vitra-A mRNA . Interes nucleus . In addie 147 nt . These rAurora-A mRNA, the protein expression level of Aurora-A in GFP-hnRNP Q1-overexpressing or hnRNP Q1 knocked-down cells was investigated. The result showed that Aurora-A is increased in GFP-hnRNP Q1-expressing cells but decreased in hnRNP Q siRNA-transfected cells  or exon1 variant (147 nt) and weakly increase the translation of exon2-containing variants . This effect is observed in nocodazole-treated GFP-hnRNP Q1-overexpressing cells. Overexpressed GFP-hnRNP Q1 increases the level of Aurora-A at G2/M phase  regulate Aurora-A mRNA 5′-UTR IRES activity remain unclear. To address these questions, the IRES activity of the four Aurora-A mRNA 5′-UTR isoforms was checked by bicistronic reporter assay, and a hairpin structure was inserted ahead of the Aurora-A mRNA 5′-UTRs to block the cap-dependent translation  to increase the RNA-binding ability.26HnRNP Q1 has been shown to bind to the AU-rich region,variants , and theR 147 nt . Meanwhixpressed . By struxpressed . The assy 147-B, . Even hnin vitro , howeverin vitro . Therefoand RBD3 . This efAurora-A 5′-UTRs  is located, has no alternation or deletion, indicating that the increased hnRNP Q may not result from gene amplification.28 It is possible that the increased hnRNP Q mRNA in tumors might be due to mechanisms involving transcriptional upregulation or post-transcriptional regulation, such as miRNA.29 The content of cancer cells might contribute additional signals in the posttranslational modification of hnRNP Q1 for its retention in the cytoplasm and its function in the translational regulation of mRNAs.26Furthermore, we found that both mRNA and protein levels of hnRNP Q1 are elevated in the tumor part of CRC compared with adjacent normal tissues .30 The reads were analyzed by using TopHat/Cufflinks31 for gene expression estimation. The levels of genes were calculated as FPKM (Fragments Per Kilobase of transcript per Million mapped reads). The reference genome and gene annotations were obtained from Human_hg19 (GRCh37.69) of Ensembl database. The data collection and analysis were performed by Welgene Biotech Co., Ltd . NGS results were submitted to GEO (Gene Expression Omnibus) with an accession number GSE76457.All the procedures were performed according to Illumina's protocol. Library constructions were estabilished by TruSeq RNA Sample Prep Kits v2 on Solexa platform . The RNA sequences were determined by using sequencing-by-synthesis technology via the TruSeq SBS Kit. For RNA-seq analysis, the sequences went through a filtering process to obtain qualified reads.in vitro transcription system  according to the manufacturer's instructions. Briefly, Aurora-A 5′-UTR was transcribed by T7 RNA polymerase (Promega) and incorporated with biotin-14-CTP  at 37 °C for 1.5 h. After treatment with RQ1 DNase (Promega), the biotin-labeled RNA probe was purified using the illustra MicroSpin G-25 Columns . Total cell lysates collected by RIPA buffer were pre-cleaned by streptavidin beads  at 4 °C for 1 h. For pull-down assay, the total cell lysates were diluted with 5X EMSA buffer  including 0.5 mg/ml tRNA, 0.7 mg/ml heparin, 100 unit/ml RNase inhibitor, protease inhibitors and biotin-labeled RNA probes. After incubating at 4 °C for 2 h, the mixtures were subjected to UV-crosslinking (120 mJ/cm2) for 5 min three times, and then streptavidin beads were added to rotate at 4 °C for 1 h. The beads were washed by the wash buffer (10 mM Hepes (pH 8), 40 mM KCl, 3 mM MgCl2, 2 mM DTT, 5% glycerol, 0.5% SDS and 2% NP-40) three times, followed by western blot analysis.The biotin-labeled RNA probe was synthesized by μg/ml) (Sigma) for different time periods. The cell lysates were collected by RIPA buffer and analyzed by western blot. The expression level of Aurora-A protein was normalized by α-tubulin and displayed the degradation curve at each time point.SW480 cells were transfected with GFP or GFP-hnRNP Q1 for 24 h and then treated with cycloheximide (200 Aurora-A 5′-UTR-pGL3 plasmids and pRL-TK vector for 12 h and then collected by 1 × passive lysis buffer (Promega). The activity of firefly and renilla luciferase was measured according to the protocol of the Dual-Luciferase Reporter Assay System (Promega) and detected by Minilumat LB 9506.The cells were co-transfected with Aurora-A 5′-UTR-phpRF plasmids for 24 h and collected by 1 × passive lysis buffer (Promega). The activity of firefly and renilla luciferase activity was measured by Dual-Luciferase Reporter Assay System (Promega) as described above.The cells were transfected with 2, 1% Triton X-100, 0.5% sodium deoxycholate, 100 unit/ml RNase inhibitor, 100 μg/ml cycloheximide). The obtained cell lysates were pre-cleaned by protein A beads at 4 °C for 1 h, and then immunoprecipitated by anti-ribosomal protein S6 antibody (Santa Cruz) for 2 h, followed by incubating with protein A beads for a further 1 h at 4 °C. The S6-IP beads were washed with RNA-IP buffer three times, and RNAs were extracted by Trizol (Invitrogen). The S6-bounded mRNAs were analyzed by RT-PCR or RT-qPCR as indicated in The cell lysates for S6-IP were collected by RNA-IP buffer (10 mM Hepes (pH 8), 100 mM KCl, 5 mM MgCl2, 2 mM DTT, 5% glycerol, 0.5% sodium deoxycholate, 100 unit/ml RNase inhibitor and protease inhibitors). The cell lysates were immunoprecipitated by anti-GFP antibody  or anti-eIF-4E antibody  for 2 h at room temperature and then incubated with 1 mg/ml tRNA pre-cleaned protein A/G beads for a further 2 h at room temperature. The bead-bound IP complex was washed with RNA-IP buffer three times and RNA was extracted by TRIsure  and analyzed by RT-PCR or real-time PCR.The cells were collected and lysed by RNA-IP buffer (10 mM HEPES (pH 8), 40 mM KCl, 3 mM MgCl32 Biotin-labeled RNA probe was synthesized using an in vitro transcription kit according to the manufacturer's instructions. (Promega). For surface plasmon resonance (SPR) analysis, the RNA probes were diluted in HBS-N buffer , 3 mM EDTA, 300 mM NaCl, 0.005% P20, in DEPC water) and injected into the sensor chip SA  at a rate of 10 μl/min for 1 min to immobilize on the chip. After immobilization, GST fusion proteins were diluted in HBS-N buffer to different concentrations, injected into the chip in a rate of 10 μl/min for 3 min, and then washed with HBS-N buffer for 7 min to detect the RNA–protein interaction on the chip by evaluating the response unit (RU). The chip was washed with regeneration buffer (3 M NaCl) to disrupt the RNA–protein interaction and rescue the probe-binding ability between each cycle of experiments.The generation of GST-hnRNP Q1 fragments and the production of GST fusion proteins were conducted as described previously.t-test was used for analyzing the result of experiments in this paper. P-value <0.05 was regarded as significant.All the experiments were repeated at least three times, and the error bar was shown as mean±S.E.M. Student's


There is general agreement among public health practitioners, academics, and policymakers that people offered health screening tests should be able to make informed choices about whether to accept. Robust measures are necessary in order to gauge the extent to which informed choice is achieved in practice and whether efforts to improve it have succeeded. This review aims to add to the literature on how to improve methods of measuring informed choice. We discuss and critique commonly-used approaches and outline possible alternative methods that might address the issues identified. We explore the challenges of defining what information should be provided about screening and hence understood by service users, appraise the use of ‘thresholds’ to define e.g. positive attitudes towards screening, and describe problems inherent in conceptualising ‘informed choice’ as a single dichotomous outcome that either does or does not occur. Suggestions for future research include providing greater detail on why particular aspects of screening information were considered important, analysing knowledge and attitude measures at an ordinal or continuous level (avoiding problematic decisions about dichotomising data in order to set thresholds), and reconceptualising informed choice as a multifactorial set of outcomes, rather than a unitary one. •There is ample scope to improve methods in studies on informed choice in screening.•Researchers could be explicit regarding how their values shape their methods.•There may be preferable alternatives to dichotomies of e.g. ‘adequate’ knowledge.•It may be overly reductive to define ‘informed choice’ as a single entity. Individan make” . To varyan make” . The lonan make” . The inean make” .an informed decision is one where all the available information about the health alternatives is weighed up and used to inform the final decision; the resulting choice should be consistent with the individual's values”  Collaboration for improving ‘decision quality’ (use event rates specifying the population and time period”). Previous studies of informed choice have been guided by both sets of recommendations , which states that screening invitees should be provided with the information that they “ option” . These rquality’ . These aons e.g. .based on a rigorous assessment of the evidence base”  screening service users. However, in a survey of older UK adults, the large majority stated a personal preference for “benefits . The combenefits ; researcbenefits . Conversbenefits . These einformed’ choice, which also requires that information is absorbed and internalised in a meaningful  way. There are also challenges in this respect: there is little apparent agreement regarding the appropriate level of ‘granularity’ to knowledge about screening. Invitees may be expected to understand a concept (“screening saves lives from breast cancer”) without necessarily being aware of the underlying statistics and their implications  and harms (in terms of overdiagnosis) are highly variable , with thhallenge .The lack of clearly superior guidelines, methods of accounting for public stated preferences, objectively appropriate levels of granularity to knowledge, and unambiguous evidence defining ‘accurate’ knowledge is reflected in review findings. There is a striking lack of consistency among studies that aim to improve informed choice in terms of what information was provided to participants and how much detail was given . There i2.2The two most common methodological approaches for setting thresholds for ‘adequate’ (or ‘good’) knowledge are to either use a standard based on observed data  a median is relative to the observed data . Second,These limitations are not specific to informed choice  but theyRelated to the challenges outlined previously, these issues are exacerbated since knowledge items are not perceived as either strictly ‘important’ or ‘not important’ but rather important to varying degrees. Although it is analytically convenient to give equal weighting to each item, this is unlikely to be a reflection of the perceptions of any given reader of a study (or participant). For example, those who consider overdiagnosis to be the main harm of screening are unlikely to assign equal importance to items that measure understanding of this concept compared with items measuring understanding of false positive results. This is not taken into account when the same score is applied to all knowledge items. These same considerations are likely to apply to measures of attitudes and intentions in that thresholds are often defined for e.g. ‘positive’ attitudes using the same problematic approaches, which also overlook differences in the relative importance of items.the terms good and poor imply an absolute standard against which knowledge is judged. It is intended that such a standard is developed in future” , rather than attempting to define and measure it based on an objective standard  is defined as a discrete event that either occurs or does not, detached from the sociocultural context. Hence, we do not take for granted that informed choice is an observable, empirical phenomenon rather than a more complex philosophical idea. Indeed, as far as we are aware, the extensive psychological research on the topic of justice itself has focused on measuring different levels of standard .2.4Per our own collective values, we believe that informed choice ought to be an integral aspect of screening policy in the UK and internationally but we also believe there should be a reappraisal of the underlying concepts and methods on which studies rely. In this section, we explore alternative approaches that we hope might overcome the previously discussed issues. We do not discuss relevant standards of methodological quality  as they apply independently of our suggested approaches.First, in order to mitigate difficulties in defining what constitutes ‘important’ information, we recommend following the example of some authors e.g. , who repGiven that selecting aspects of information to provide and aspects of knowledge to assess requires researchers to draw from their own values, we also suggest they are as explicit as possible about why selected topics were considered important. For example, if researchers select items based on particular guidelines, it is worth clarifying the rationale for using that set and the process used to map specific recommendations on to knowledge items. Similarly, it would be informative to explain how items were selected based on input from (prospective) screening participants, if applicable. Researchers could also clarify whether any perceived practical or ethical considerations meant the final set of knowledge items did not fully reflect guidelines or services users' stated preferences, and highlight limitations of the selected approach . Finally, research evidence used to determine ‘correct’ responses to knowledge items should be cited clearly e.g. . These mWe have also argued that dichotomising measures of knowledge, attitudes, and intentions is difficult to justify on empirical or theoretical grounds. Subjective and arbitrary thresholds may be addressed by taking into account the natural properties of the data . Analyses that reflect this are likely to be more robust and would also address the additional problem that information is lost when data are dichotomised . It may state of uncertainty about the course of action to take” (I feel pressure from others in making this decision”). These might be used in addition to or instead of standard components of informed choice. Furthermore, although we have focused on a particularly common method of measuring informed choice, there are other factors that may be considered relevant and are not encapsulated by knowledge, values, and behaviour . In theito take” : decisioWe also suggest that research could be improved if the three components of informed choice were evaluated individually, rather than attempting to unify them into a single overall ‘construct’. A more wide-ranging and less reductive conception of informed choice might also lead researchers to consider novel hypotheses. For example, since screening invitees are generally asked to consider information in the context of complicated social and cultural dynamics between themselves and a healthcare provider , studiesThe following example illustrates some practical ways in which these methods might benefit policymakers and healthcare providers: a healthcare provider might commission a randomised controlled trial to compare a new information leaflet that aims to better inform invitees about the risk of overdiagnosis in breast screening cf  with theIn closing, we acknowledge that following our suggestions would have some limitations. First, reporting more outcomes may raise potential statistical issues around testing multiple hypotheses but we would argue that these are well-recognised and relatively straightforward to address in comparison to the more problematic conceptual issues that we believe would likely be resolved. Second, undertaking more complicated analyses and interpreting a broader range of outcomes may be more challenging and time-consuming than other methods. We would counter that they are likely to be more useful to policymakers and healthcare providers in the long-term if they offer more validity. However, a potential threat to this usefulness is if there is significant divergence between researchers' conceptions of (e.g.) the information they believe to be important and the views of service users and policymakers. This may lead to study results being irrelevant or impossible to compare. However, we are not suggesting an entirely individualistic approach to defining important topics but one in which service users, healthcare providers, policymakers, academics, and other stakeholders continue to shape the views of one another, which is likely to minimise disagreement. We would also argue that the methods we have suggested are not creating issues themselves but rather adding transparency around issues that are inherent in the topic.We have focused on quantitative methods of studying informed choice because we believe there are significant benefits to generating information that is readily interpretable and actionable. Although we hope that quantitative approaches can still be justified, one might extend our arguments to the conclusion that informed choice is ultimately too subjective to be amenable to this approach. This might imply that an even more abstract conceptualisation may be warranted. For example, policymakers may promote the value of informed choice as a cultural norm among practitioners, the effects of which might suit qualitative methods of assessment. We offer our suggestions as a contribution to future discussions for improving the measurement of informed choice.3We have discussed some important limitations of commonly used approaches to measuring informed choice, which suffer from a lack of agreed standards regarding which aspects of knowledge are important, subjective or inappropriately defined thresholds for dichotomising key components, and an overly reductive conceptualisation. We suggest some alternative approaches that may be more appropriate. Instead of attempting to define informed choice as either occurring or not occurring, we suggest reporting detailed results using individual items, explicit descriptions of how information and items were selected, avoiding the use of thresholds, greater use of regression methods to explore associations between components, and more wide-ranging relevant measures. We hope our suggestions will foster discussions on how informed choice research can be developed, as this will allow more valid auditing of the extent to which informed choice is achieved in practice, and lend greater weight to comparisons of interventions that aim to improve screening.Cancer Research UK awarded to Prof Jane Wardle (C1418/A14134). Cancer Research UK was not involved in the writing of the manuscript; or in the decision to submit for publication.The current article was supported by a programme grant from AG conceived this review. All authors participated in drafting and critical revision of the manuscript, and approved the final version.No conflicts of interest have been declared.Transparency document.


Coding region determinant-binding protein (CRD-BP) binds to the 3’-UTR of microphthalmia-associated transcription factor (MITF) mRNA to prevent its targeted degradation by miR-340. Here, we aim to further understand the molecular interaction between CRD-BP and MITF RNA. Using point mutation in the GXXG motif of each KH domains, we showed that all four KH domains of CRD-BP are important for their physical association with MITF RNA. We mapped the CRD-BP-binding site in the 3’-UTR of MITF RNA from nts 1330–1740 and showed that the 49-nt fragment 1621–1669 is the minimal size MITF RNA for binding. Upon deletion of nts 1621–1669 within the nts1550-1740 of MITF RNA, there was a 3-fold increase in dissociation constant Kd, which further confirms the critical role sequences within nts 1621–1669 in binding to CRD-BP. Amongst the eight antisense oligonucleotides designed against MITF RNA 1550–1740, we found MHO-1 and MHO-7 as potent inhibitors of the CRD-BP-MITF RNA interaction. Using RNase protection and fluorescence polarization assays, we showed that both MHO-1 and MHO-7 have affinity for the MITF RNA, suggesting that both antisense oligonucleotides inhibited CRD-BP-MITF RNA interaction by directly binding to MITF RNA. The new molecular insights provided in this study have important implications for understanding the oncogenic function of CRD-BP and development of specific inhibitors against CRD-BP-MITF RNA interaction. Coding region determinant-binding protein , also known as IMP1 (human), belongs to a highly conserved family of RNA-binding proteins called VICKZ; all which possess two N-terminal RNA-recognition motifs (RRMs) followed by four C-terminal KH [hnRNP (heterogeneous nuclear ribonucleoprotein) K-homology] domains . Two animyc mRNA, and its ability to influence c-myc mRNA stability was demonstrated in cell-free and cell line models  UTP (3000 Ci/mmol). Following incubation, 3 units of RNase-free DNase I (Promega) were added, and the reaction was further incubated for 10 min at 37°C. Upon addition of 10 μl Stopping dye , the entire sample was electrophoresed on a 8% polyacrylamide/7M urea gel. Bands containing internally-radiolabeled RNA and determined to be the correct size, were excised, and eluted with elution buffer  at 45°C for 6 hours. The purified, radiolabeled RNA was then phenol/chloroform extracted followed by ethanol precipitation. Specific activity of the RNA was then determined by scintillation counting.The human MITF  and β–TrCP1  cDNAs purchased from Thermo Scientific Open Biosystems  were used as templates for the generation of PCR-amplified DNA fragments. The different sets of forward and reverse primers used to amplify the different regions of MITF cDNA are shown in in-vitro transcription reaction contained 5 μg of gel purified DNA template, 10 μL 10 x T7 buffer , 5 μL each of 100 mM ATP, CTP, GTP, and UTP, 40 units RNasin, 5 units of T7 RNA polymerase to a total volume of 100 μL. The reaction was incubated at 37°C overnight followed by treatment with DNase I to remove the DNA template. Phenol-chloroform extraction was performed to remove protein impurities followed by passing through G-50 spin column to remove unincorporated nucleotides. RNA was ethanol precipitated and re-suspended in DEPC-treated water. Purity of RNA was checked by visualization on 2.5% agarose gel, and their concentration was determined using spectrophotometer.For the synthesis of unlabeled MITF RNA nts 1550–1740 and GLI1 RNA nts 346–382 for use in competitive electrophoretic mobility shift assay (EMSA), the following reactions and procedure were taken. Each 32P]-labelled MITF RNA nts 1550–1740 was added to the reaction. This was followed by the standard EMSA protocol. The molar excess concentrations of antisense oligonucleotides MHO-1 to MHO-8, DD7 or GLI1 RNA nts 346–382 over the [32P]-labelled MITF RNA are shown in the competitive EMSA studies.The electrophoretic mobility shift assay (EMSA) was conducted in essentially the same manner as previously described ,22. EMSA32P-labelled MITF 1550–1740 RNA probe to separate tubes containing 2 μg of DNA oligonucleotides (MHO-1 or MHO-7) or 2 μg yeast tRNA as control. The nucleic acid samples were precipitated and resuspended in 10 μl of Hybridization Buffer III (Ambion RPA III kit) followed by denaturation at 95°C for 3 min, and hybridization overnight at 42°C. Three 150 μl RNase H solutions containing 7.5 U of RNase H (New England Biolabs) in 1 X RNase H buffer were prepared and mixed with each hybridization reaction and incubated at 37°C for 1 hour, followed by heat inactivation at 65°C for 20 min. Digested RNA was then precipitated at -20°C for 1 hour after adding 225 μl of RNase Inactivation/precipitation III solution (Ambion RPA III kit), followed by 30 min centrifugation at 10,000 x g. RNA pellets were dried and suspended in 10 μl of Gel Loading Buffer II (RPA III kit) and fully denatured by heating at 95°C for 3 min. RNA samples were then resolved by gel electrophoresis on an 8% denaturing (7 M urea) polyacrylamide gel and exposed overnight to phosphor screen before visualization using the Cyclone Plus phosphorimager system (PerkinElmer).DNA-RNA complexes were first generated by adding 20,000 cpm of 5’-TAA AAA TCT CAA ATC AAG TTT C/36-FAM/-3’); MHO-7_FL (5’-ATT CAG TTC ACA GAT ACT GCA C/36-FAM/-3’); MHO-6_FL (5’-ATA CTG CAC ACT TAT TTG TAC A/36-FAM/-3’). As a positive control, we used 39-nt fluorescent-labeled CD44 (FL-CD44) RNA (5’- AAA UUA GGG CCC AAU UAA UAA UCA GCA AGA AUU UGA UCG /36-FAM/ -3’) which is known to bind to CRD-BP. All fluorescein-labeled nucleic acids were synthesized by IDT Inc. For assessment of binding to CRD-BP, the following reaction mixture was used: 4 μl of binding buffer , 7 μl recombinant CRD-BP (0–1000 nM), 5 μl fluorescein-labeled nucleic acids (10 nM), and 3 μl sterile water. The mixtures were added to 384 Microfluor Microtiter plate (Thermo Electron) and incubated at 37°C for 30 min prior to plate reading. For assessment of binding to MITF RNA 1550–1740, the following mixture was used: 4 μl Hybridization Buffer III (Ambion RPA III kit), 7 μl MITF RNA 1550–1740 (0–1000 nM), and 8 μl fluorescein-labeled nucleic acids (10 nM). The mixtures were incubated at 42°C for 4 hours to allow nucleic acid binding. Plates were read at excitation wavelength 485 nm and at emission wavelength 528 nm using Gen5 software in Synergy 2 .The following fluorescein-labeled oligonucleotides were used: MHO-1_FL , suggesting that the control variants have slightly higher affinity for MITF RNA. Surprisingly, the KH single variants, KH2, KH3 and KH4, had abolished ability to bind MITF RNA  against MITF RNA , and subin vitro by binding to the MITF RNA. In addition to examining RNAs, we also show that all four KH domains are required by CRD-BP to bind MITF RNA. Taken together, this study has provided new insights into the CRD-BP-MITF RNA interaction. Such information will inevitably aid in future development of inhibitors of CRD-BP function.In conclusion, we have characterized the interaction between CRD-BP and MITF RNA nts 1550–1740 and mapped the MITF RNA region down to 49 nts in size, which still has affinity for CRD-BP. Our deletion studies further confirmed the importance of nts 1621–1669 within the 3’UTR of MITF RNA in binding to CRD-BP. Furthermore, we revealed two antisense oligonucleotides that can block the CRD-BP-MITF RNA interaction S1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.


CT引导下穿刺活检（computed tomography-guided needle biopsy, CTNB）是诊断肺癌最常用的一种方法，具有诊断准确性高及并发症少的特点。现有的研究表明CTNB可引起肿瘤的针道转移，然而极少有研究关注CTNB对远处转移和远期生存的影响。本研究旨在探讨术前的CTNB对Ⅰ期-Ⅱ期非小细胞肺癌（non-small cell lung cancer, NSCLC）远处转移和生存的影响。Cox回归分析法和Kaplan-Meier分析法进行生存分析。研究对象为1, 234例Ⅰ期-Ⅱ期NSCLC术后患者，其中113例术前行CTNB，1, 121例术前未经任何活检。应用倾向性评分匹配方法平衡两组间的临床特征，经配对后纳入后续统计检验共226例。并进一步应用P=0.032），而两组间总生存无统计学差异（P=0.086）。在Ⅰ期-Ⅱ期NSCLC患者中，术前的CTNB组无远处转移时间（distant metastasis free survival, DMFS）显著短于无活检组（CT引导下穿刺肺活检可能促进早期肺癌的远处转移，但不影响总生存。


Type 3 RNA polymerase III (Pol III) promoters are widely used for the expression of small RNAs such as short hairpin RNA and guide RNA in the popular RNAi and CRISPR-Cas gene regulation systems. Although it is generally believed that type 3 Pol III promoters use a defined transcription start site (+1 position), most man-made promoter constructs contain local sequence alterations of which the impact on transcription efficiency and initiation accuracy is not known. For three human type 3 Pol III promoters , we demonstrated that the nucleotides around the +1 position affect both the transcriptional efficiency and start site selection. Human 7SK and U6 promoters with A or G at the +1 position efficiently produced small RNAs with a precise +1 start site. The human H1 promoter with +1A or G also efficiently produced small RNAs but from multiple start sites in the −3/−1 window. These results provide new insights for the design of vectors for accurate expression of designed small RNAs for research and therapeutic purposes. Small RNAs play regulatory roles in diverse intracellular processes in both eukaryotes and prokaryotes. In addition, small RNA molecules have been exploited as powerful tools for biomedical research and therapeutic purposes. This includes small interfering RNA (siRNA) or short hairpin RNA (shRNA) in RNA interference (RNAi) experiments, guide RNA (gRNA) in clustered regularly interspaced palindromic repeats (CRISPR)-Cas applications and RNA molecules such as aptamers, ribozymes, and antisense RNA.The accurate expression of designed small RNAs is important for the proper and specific execution of their function. For instance, for shRNA molecules, the sequence precision is critical because the stem-loop structure is processed by the Dicer endonuclease that measures ∼21-nucleotides (nt) from the 5′ terminus and length variation at this end will yield a different RNA duplex with different silencing activity and specificity.Pol III promoters are efficient in transcribing high levels of small non-coding RNAs and promoters such as 7SK, U6, and H1 are therefore widely used.Acc65I restriction enzyme site in the wild type (wt) promoter sequence (GGTACC underlined in http://www.ncbi.nlm.nih.gov/) indicated that all use +1G (results not shown).The human 7SK promoter initiates transcription of the abundant 7SK nuclear RNA that represses Pol II transcription elongation by binding to the elongation factor P-TEFb.To examine whether the identity of the +1 nt affects 7SK promoter activity, we made all four possible +1 variants in two reporter constructs, 7SK-Luciferase (7SK-Luc) and 7SK-N44, a manmade sequence that produces a 44-nt transcript that lacks stable RNA structure . The forTo distinguish between transcription versus post-transcriptional effects, we next used the 7SK +1 variants to transcribe the small unstructured N44 sequence . Total cWe next wanted to investigate whether nucleotide changes at the +1 position also affect the transcription start site selection. 5′ rapid amplification of cDNA ends (5′ RACE) is a highly sensitive method for mapping the 5′ end of individual RNAs and was used for mapping Pol III transcription start sites. We previously mapped the transcription start site profile of the H1 promoter in the pSUPER construct.BamHI restriction enzyme site that facilitates cloning , which is an essential component of the spliceosome.As described for the 7SK promoter, we used these U6 variants to drive Luc expression and N44 transcription. Consistent patterns were observed in the Luc assays for all three sets of U6 promoter constructs: The variants with A or G at the +1 or +2 position are stronger than the corresponding T and C constructs . NortherNext, we used GeneScan to map the transcription start sites . The staThe H1 promoter controls the transcription of H1 RNA, the RNA component of the nuclear RNase P enzyme.BgIII restriction enzyme site for easy cloning purposes . We measured increased anti-Luc activity, especially for the −2/−3 construct compared with the standard +1 construct, highlighting the importance of start site knowledge. A complicating factor is that different H1 constructs are in use, and commercial sources have generated modified H1 constructs with convenient restriction sites for cloning purposes.A previous study indicated that a yeast internal Pol III promoter prefers purines at the transcription initiation site.The three Pol III promoters analyzed in this study have been widely used for synthesis of small RNAs, but some reports indicated that the U6 promoter can also drive the expression of a Luc reporter gene, resulting in a translation-competent mRNA.The psiRNA-h7SK hygro G1 (Invivogen), pSilencer 2.0-U6 (Ambion), and pSUPER (OligoEngine) vectors that contain different human Pol III promoters were used. The actual sequences of the 7SK, U6 and H1 promoters used in the study are provided as Supplemental material. The N44 sequence (5′-ACCATGGAAGTGAAGGGGCAGTAGTAATATACCGGTGATATCTT-3′) with nucleotide variation around +1 was inserted into these three vectors.To construct the anti-Luc CRISPR-Cas9 system, the CRISPR plasmid px458 (Addgene plasmid #48138) was used as backbone. We designed gRNAs targeting HIV Gag (5′-GCTACCATAATGATGCAAAG-3′) and the Firefly luciferase reporter (5′- GTGAACTTCCCGCCGCCGTT-3′). All H1-gRNAs were synthesized by Integrated DNA technology (IDT) and cloned into px458 by Gibson cloning according to the protocol (New England Biolabs). All constructs were verified by sequencing using BigDye Terminator v1.1 Cycle Sequencing Kit (ABI).Human embryonic kidney (HEK) 293T cells were cultured in Dulbecco's modified Eagle's medium  supplemented with 10% fetal calf serum (FCS), penicillin (100 U/ml) and streptomycin (100 μg/ml). The cells were trypsinized and seeded one day before transfection.5 cells was seeded in 24-well plates. Pol III promoter-based pGL3 plasmids were titrated , and 1 ng of Renilla luciferase plasmid (pRL) was co-transfected into HEK293T cells with Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. For measuring the anti-Luc activity of the gRNAs, equal amounts (200 ng) of px458 plasmid were co-transfected with 200 ng pGL3-control and 2 ng pRL plasmid. Two days post-transfection, luciferase activity was measured with the Dual-Luciferase Reporter Assay System  according to the manufacturer's protocol. The ratio of Firefly to Renilla luciferase was used for normalization of the transfection efficiency. Three independent transfections were performed, each in duplicate. The resulting six values were corrected for between session variations as described previously.0.5 ml DMEM/10% FCS with 1.5 × 106 HEK293T cells per 25 cm3 flask were transfected with equimolar quantities (5 μg) of N44 constructs using lipofectamine 2000 (Invitrogen). Total cellular RNA was extracted two days post-transfection with the mirVana miRNA isolation kit (Ambion). The RNA concentration was measured with NanoDrop 2000 (Thermo Fisher Scientific). 5 μg of total RNA was electrophoresed in a 15% denaturing polyacrylamide gel . [γ-32P]-labeled decade RNA marker (Life Technologies) was run along-side for size estimation. To check for equal sample loading, the gel was stained in 2 μg/ml ethidium bromide for 20 minutes and visualized under UV light. The RNA in the gel was electro-transferred to a positively charged nylon membrane  and cross-linked to the membrane using UV light (1200 μJ × 100). Locked nucleic acid (LNA) oligonucleotides (Pol47: 5′-ATTACTACTGCCCCTTCAC-3′) were 5′ end-labeled with the kinaseMax kit (Ambion) in the presence of 1 μl [γ-32P]-ATP . Sephadex G-25 spin columns (Amersham Biosciences) were used to remove the unincorporated nucleotides. The membrane was incubated in 10 ml ULTRAhyb hybridization buffer (Ambion) at 42°C for 30 minutes, after which the labeled LNA oligonucleotides were added. After overnight hybridization at 42°C, the blot was washed twice for 5 minutes at 42°C with 2 × SSC/0.1% SDS and twice for 5 minutes at 42°C with 0.1 × SSC/0.1% SDS. The signals were captured by the Typhoon FLA 9500  and quantified using ImageQuant. Two independent transfections and Northern blottings were performed.Northern blotting was performed as described previously.5 μg of total cellular RNA of N44-transfected cells was reverse transcribed with the First-Strand cDNA Synthesis Kit (ThermoScript RT-PCR system). The 6-FAM labeled primer (5′-GATATCACCGGTATATTA-3′) complementary to the 3′ end of the N44 RNA sequence was used for primer extension. After incubation with RNase H, the cDNA was concentrated by ethanol precipitation and mixed with 1.5 μl Rox 500 Size Standard and run on an ABI PRISM 3010 XL Genetic Analyzer (Applied Biosystems) using default parameters. All data were analyzed using GeneMapper® software v4.0 (Applied Biosystems) and then manually corrected based on fragment size to generate the N44 transcription start site profile.


Along conventional dual labelled probes (DLP), alternative primer and probe chemistries, including Zip Nucleic Acids (ZNAs), Locked Nucleic Acids (LNAs), and Scorpions®, were compared with two RNA template types: i) total genomic RNA extracted from cell cultured influenza A and ii) a synthetically prepared RNA transcript (In vitro transcribed RNA).Compared to other PCR technologies, digital PCR is a potentially highly accurate approach for the quantification of nucleic acid fragments. This study describes the impact of four experimental factors, namely primer and probe chemistry, PCR amplification target, duplexing, and template type, on the measurement results obtained by reverse transcription digital PCR (RT-dPCR) of viral RNA using While apparently duplexing or a different PCR target choice did not have a significant influence on the estimated RNA copy numbers, the impact of the choice of primer and probe chemistry and template type differed significantly for some methods. The combined standard uncertainty of the dPCR analysis results has been assessed, taking into account both the repeatability and the intermediate precision of the procedure.Our data highlight the importance of dPCR method optimisation and the advantage of using a more sophisticated primer and probe chemistry, which turned out to be dependent on the template type. Considerations are provided with respect to the molecular diagnostics of viral RNA pathogens, and more specifically, for precise quantification of RNA, which is of tremendous importance for the development of RNA calibration materials and the qualification of these calibrants as certified reference materials. Therefore, dPCR uses the same primers and probes as the widely applied qPCR, but has been reported to be capable of higher sensitivity and precision due to the underlying principle of limiting dilution, whereby the sample is diluted and partitioned into many separate reaction partitions (chamber-based dPCR) or droplets (droplet-based dPCR) before amplification ® probe, are widely employed for (RT-) qPCR and (RT-) dPCR. In recent years, an increasing number of alternative PCR chemistries have become available. These include Locked Nucleic Acids (LNA), Zip Nucleic Acids (ZNA), and Scorpions®, among others. Several studies demonstrated that the use of alternative primer and/or probe chemistries may offer improved assay sensitivity Another influencing factor could be the choice of the primer and probe system. Conventional dual labelled probes (DLP) with a fluorophore and a quencher, such as a Taqman22.12.1.1In vitro transcribed RNA was synthesised in-house, using the pGEM®-T easy plasmid vector containing an insert, covering the entire segment 7 (M gene) of influenza A. The insert originated from RNA extracted from influenza A virus A/Aichi/2/68 (H3N2) purchased from the ATCC® Collection . SuperScript® VILO™ Master Mix  was used for cDNA synthesis. The insert was generated using High Fidelity Platinum® PCR SuperMix (Invitrogen) and previously published primers Bm-M-1 and Bm-M-1027R ®-T Easy Vector System II  was carried out according to the manufacturer’s recommendations. Plasmid DNA was extracted from a 5 ml Luria-Bertani (LB) broth culture (grown overnight in a shaking incubator at 37 °C) using The Wizard® Plus SV Minipreps System (Promega) and following the manufacturer’s protocol. The identity of the insert was confirmed by sequencing of both DNA strands . In vitro transcription was carried out with the RiboMAX™ Large Scale RNA Production System and T7 polymerase (Promega) using SalI (Promega) − linearised plasmid as a template. The transcript was purified using SV Total RNA Isolation System (Promega) according to the producer's protocol, which included treatment with DNase I to remove unwanted DNA from the RNA preparation. The size, purity and integrity of the IVT RNA were confirmed by analysis on the Agilent 2000 Bioanalyzer using the RNA 6000 Pico kit . Purified IVT RNA was diluted in the RNA Storage Solution  and 50 μl aliquots were stored at −70 °C. The concentration of IVT RNA was assessed by spectrophotometry  to be 1.83 × 1010 copies/μl. Based on the sequencing results and the position of the T7 promoter, the size of the expected RNA molecule was assessed to be 1147 bp.2.1.2® Viral RNA Mini kit , according to manufacturer's protocol, with minor modifications concerning the elution of the RNA. Samples were eluted in two steps, each using 120 μl RNA Storage Solution (Ambion®). Extracted RNA samples were pooled and gently mixed to obtain a homogeneous solution. Twenty and fifty μl aliquots were stored at −70 °C. The concentration of the total genomic influenza RNA was determined by dPCR using 12.765 digital arrays on the BioMark™ HD System  according to the published CDC protocol targeting a conserved sequence of the influenza A matrix gene 6 copies/μl and relative standard deviation (RSD) of 2.2%.Genomic RNA was prepared from cell cultured influenza A virus strain A/Aichi/2/68 (H3N2) propagated in St Georges Hospital, London, and extracted using QIAamp2.22.2.1® and LNA primers and probes were designed using the online tool OligoArchitect™ . All primers and probes were synthesised and HPLC-purified by Sigma UK, except for ZNAs which were synthesised by Sigma USA. Single-use aliquots of oligonucleotides, reconstituted in nuclease-free water (Promega) were stored at −20 °C. Primer and probe information is included in In total, seven different primer and probe chemistries were applied in this study and are indicated by a specific code in 2.2.2®) and run in triplicate (extracted gRNA) or quintuplicate (IVT RNA). The position of different experiments on the digital array was randomised over three different days. The same sample dilution was used for all the methods on individual days to allow for direct comparison. The digital array was primed and loaded according to manufacturer's protocol. Thermal cycling conditions were: 50 °C for 30 min for reverse transcription, 95 °C for 2 min for denaturation and inactivation of RTase, followed by 45 PCR cycles at 95 °C for 15 s and 55 °C for 30 s ®), constituting the no-template control, was included in each experiment. Also a negative control, sonicated human gDNA (Cambio) at 25 ng/μl was analysed to check for unspecific reactions. The analysis was performed utilising the Fluidigm dPCR software version 4.1.2 to assess the concentration by counting the number of positive partitions (H) out of the total number of partitions (C) from which the Poisson distribution was used to estimate the average number of DNA copies per partition (λ) via λ = ln(1-H/C) dPCR experiments were performed using the 37K IFC Digital Arrays of the BioMark platform (Fluidigm) and the RNA UltraSense™ One-Step Quantitative RT-PCR System (Invitrogen). Quantification of the RNA was done under intermediate precision conditions (independent runs performed on different days) with samples diluted gravimetrically in RNA storage solution buffer (Ambion®). When extracted genomic RNA (gRNA) of influenza A was used as a template, nine different methods were evaluated. In addition to the six methods mentioned above, which are all amplifying particular fragments of the M gene, the extracted gRNA was also quantified with an H3- primer and probe set targeting another gene of the influenza A virus, the haemagglutinin (HA) encoding gene (DLP-HA gene) vs. HEX) Six methods were applied for the analysis of influenza A IVT RNA, consisting of the established RT-PCR methods from CDC 2.2.3Conventional real-time PCR was carried out to determine the optimal annealing temperatures for different primer and probe sets compared in this study. Extracted genomic RNA was diluted volumetrically and used as a template together with the RNA UltraSense™ One-Step Quantitative RT-PCR System (Invitrogen).® LE Agarose, Cambrex Bio Science Rockland, Inc., Rockland, ME, USA) and visualised by gel staining with GelRed™ Nucleic Acid Gel Stain  and electrophoresis.The cycling conditions were: 50 °C for 30 min, 95 °C for 2 min, followed by 45 cycles at 95 °C for 15 s and varying annealing temperatures for 30 s ®). The extracted genomic RNA was diluted 1: 500 to fall within the optimal working interval for dPCR quantification, which is 200–700 positive chambers per panel, corresponding to 230–1900 copies/panel or a λ- value between 0.3 and 2.5 after Poisson correction To evaluate the influence of different primer and probe concentrations on measurements with the 37K array of the BioMark dPCR system, 8 μl reaction mixtures were used, comprising 3 μl RNA, 1.6 μl RNA UltraSense™ One-Step Quantitative RT-PCR master mix, 0.4 μl 20 x GE sample loading reagent, 0.4 μl RT/Taq mix, 0.16 μl ROX dye and either 0.25 μl, 0.4 μl or 0.6 μl of primers and probes according to the respective primer and probe concentrations investigated. The volume was brought to 8 μl by the addition of nuclease-free water  causes the null hypothesis to be rejected, further testing has been performed to determine which pairs of means are significantly different. This was done by Tukey's Honest Significant Differences (HSD), a Post-hoc test, where group means are compared pairwise to determine whether the difference between the pair of means is significant. The two means are significantly different if the statistic q-value is larger than the critical q-value , which is based on the probability of error, α (0.05), the number of groups, r, and the degrees of freedom (dfw) of MSwithin, calculated from ANOVA. This value can be obtained from a table of the Studentized range q distribution. The statistic q- value is calculated according to Eq. MSwithin mean square within a run from an ANOVAn the number of independent replicates per dayThe statistical analysis of the measurement data was performed by calculation of descriptive parameters such as mean value, standard deviation (SD) and relative standard deviation (RSD), and by single-factor analysis of variance (ANOVA) in Microsoft Excel 2010. The ANOVA is testing the null hypothesis which assumes that all of the group means in that test are the same. When a significant result from the ANOVA test (srep) and the intermediate precision  were derived according to Eq. MSbetween mean square between runs from an ANOVA.For each method, the repeatability . As specified in the methods chapter above, samples were subjected to 8 different annealing temperatures in the range of 50 °C–65 °C and final reaction products were visualized by gel electrophoresis. Fig. S2 shows the gel images of this experiment for the primer and probe sets (number 1–6) tested. Visual inspection of the gel images showed single amplification products for all sets, run at different annealing temperatures, except for locked nucleic acid (LNA) primers and probe. Although an optimal annealing temperature of 65 °C for LNA probes has been previously reported to be useful for more sensitive DNA detection p < 0.001) . According to the data obtained, a final concentration of primers and probes of 0.8 μmol/l and 0.2 μmol/l, respectively, was selected for all methods, except for the ZNA method, which will be used at a primer concentration of 0.5 μmol/l and probe concentration of 0.3 μmol/l.A second optimisation experiment was performed using dPCR to determine the optimal concentration of the different primer and probe sets used. For none of the methods, significant differences between the various primer and probe concentrations could be observed, except for the ZNA primer and probe set. The highest copy numbers per microliter were obtained for the ZNA primer: probe concentration of 0.5 μmol/l: 0.3 μmol/l, while only 14–28% of the RNA copies could be recovered when a higher primer concentration was used (3.23.2.1in vitro transcribed RNA fragment (IVT RNA) and total genomic RNA extracted from Influenza A virus. 10 copies/μl), followed by ZNA (1.72 × 1010 copies/μl), the conventional DLP − GRAM method (1.58 × 1010 copies/μl), and Scorpions® (1.57 × 1010 copies/μl) technology. According to results from the HSD Tukey's test, the difference between the average RNA copy numbers obtained with the LNA and ZNA methods, targeting the same region of the Influenza A matrix gene were not statistically significant, neither were the differences between Scorpions® and DLP – GRAM The influence of different primer and probe chemistries on RT-dPCR quantification was investigated for two RNA template types, a small (1147 bp) LNA method is associated with higher estimates of concentration when measuring IVT RNA, is in accordance with previous studies, demonstrating superior performance of LNA primer and probes in terms of efficiency and sensitivity The finding that the Zip Nucleic Acids (ZNA) method was developed. ZNA are oligonucleotides conjugated with spermine residues as cationic units to reduce the negative charge and favour hybridisation with a complementary sequence by clipping the strands together like a zipper, hence their name Zip Nucleic Acids Another approach to improve the nucleic acid binding affinity is to decrease the electrostatic repulsion between negatively charged nucleic acid strands. Based on this electrostatic interaction mechanism the ®,Scorpions a method wherein primer and probe are combined into a single molecule. A Scorpion consists of a specific probe sequence held in a hairpin loop conformation by complementary stem sequences on both ends of the probe. The probe contains a 5′-end reporter dye and a 3′-end quencher dye directly linked to the 5′-end of a PCR primer via a PCR blocker. The PCR blocker prevents that the PCR primer is further extended, which could lead to the opening of the hairpin loop in the absence of the specific target and to the detection of non-specific PCR products ® chemistry is kinetically favourable and highly efficient due to the direct binding of the probe to the target sequence, ensuring that each probe has a target in the near vicinity. Enzymatic cleavage is not required, which improves the PCR reaction time significantly in comparison with conventional dual labelled probes. There is a direct relationship between the number of amplicons generated and the emitted fluorescence signal ® primers when compared to dual labelled probes due to its unimolecular probing mechanism, our data did not confirm this expected higher sensitivity compared to the conventional DLP method targeting the same region of the M gene, DLP-GRAM  needs to be assessed. When RT-dPCR is applied to assign values to reference materials, a bias should be as low as possible or ideally eliminated. It is known that RT-dPCR introduces a larger variability into the measurement results than dPCR on its own 3.2.210 and 1.14 × 1010 copies/μl, respectively and extracted gRNA: 1.09 × 106 and 1.10 × 106 copies/μl, respectively).Besides the influence of the dPCR template type and the primer and probe chemistry, the choice of the target may also have an effect on dPCR measurements. When comparing the results obtained with the classical dual labelled primer and probes, targeting different regions of the same influenza A matrix gene, DLP- FAM and DLP – HEX versus DLP-GRAM, or even of a different gene (DLP-HA gene), no significant difference could be found for the extracted gRNA template . However3.2.3Larger variations were observed between RNA concentrations obtained with different primer and probe chemistries when an IVT RNA fragment was targeted (RSD of 18%) compared to the extracted genomic RNA template (RSD of 9%). The results from the HSD Tukey's test revealed important differences between the two RNA template types. For IVT RNA, twelve out of fifteen (resembling 80%) method combinations were significantly different, while only six out of thirty-six (resembling 17%) of the combinations for extracted genomic RNA were found to provide significantly different RNA copy number concentrations (Table S3). The significant increase in RNA copy numbers observed when the ZNA primers and probe were used in comparison with its unmodified counterpart, DLP – FAM, was more than 32% for IVT RNA, while this large difference was mitigated to 14% when extracted gRNA was used as a template. Further, a 36% difference was measured between the highest and lowest measurement results for IVT RNA, while the difference between the maximum and minimum values obtained for extracted gRNA was only 16%.in vitro synthesis and not for the longer, more complex genomic RNA isolated from influenza A virus cell culture. A possible explanation could be that the smaller IVT RNA is less stable than the gRNA template. Higher affinity and, consequently, more efficient hybridisation of the modified oligonucleotides, such as ZNA or Scorpions, could result in faster reaction kinetics allowing for generation of cDNA before any degradation of the RNA sample occurs. Further studies are needed to confirm this hypothesis.This interesting different behaviour of two nucleic acid template types has also been described for quantitative dPCR data of DNA, for which the more complex secondary structure of the genomic DNA and the associated higher molecular dropout was seen as an important factor for the greater variance observed with purified genomic DNA as template 3.2.4It should also be noted that there was almost no effect of duplexing. During duplexing the measurement methods targeting the M gene and HA gene were combined in a single reaction and compared to the detection of the respective genes in separate methods , sip. Five replicate measurements were performed per method for IVT RNA and this in three independent runs, each on a different day. For gRNA extracted from virus culture, measurements were done in triplicate on each of 3 different days. Uncertainties were calculated based on srep and sip according to the equations as given above. No bias related uncertainty has been included.When estimating the combined standard uncertainty for the precision of a measurement procedure, it is necessary to assess the repeatability (within-run), When measurements were performed on IVT RNA, relative combined standard uncertainties were on average 3.4%, with all of the methods having uncertainties <5% except for the method using a conventional dual labelled probe and fluorophore FAM (DLP-FAM), for which 6.3% was estimated. The majority of this larger standard uncertainty could be attributed to an increased value of 10% for the intermediate precision (The methods with the strongest PCR response for IVT RNA (ZNA and LNA) had a comparable combined standard uncertainty of about 3%. These low uncertainty values could be confirmed when extracted gRNA was used as a template, resulting in uncertainties from 2.9% (DLP – GRAM) to 6.8% for the DLP – HA gene method. This slightly higher uncertainty value of the DLP – HA gene method was due to an intermediate precision of 11.4%, whereas the intermediate precision for the DLP – GRAM method was much better with 1.4%. The average relative standard uncertainty for measurements on the extracted genomic RNA template was found to be 5%. Overall, the standard uncertainty of the values measured, independent of the method or sample template evaluated, was ≤6.8%, which illustrates the ability of RT-dPCR to quantify viral RNA in a precise and reproducible manner, without the need for calibration with a matched nucleic acid template.4Although the theory of dPCR assumes that each target nucleic acid molecule present in a partition of the BioMark dPCR chip (Fluidigm) will undergo successful amplification, this study and many others have shown that more careful method design and optimisation are required to obtain an accurate quantitative result This study has also shown that the replacement of the fluorophore from FAM- to HEX- did not have any impact on the obtained RNA copy numbers. Further, no effect on the RNA concentration has been found for the simultaneous detection of two different genes in duplex test format, compared to the result of their single-plex partners. However, targeting different amplification regions may result in differences in RNA or DNA concentration, which could be of major importance, depending on the application of the measurement results.In studies where RNA reference materials intended for RT-qPCR calibration would be characterised by RT-dPCR, approaches as applied in this study should be taken into account to ensure value assignment with minimal negative bias due to amplification drop out. It is important to emphasise that, based on the limited studies carried out so far, and in line with the statement of Josefsen et al. Overall, this study demonstrates that RT-dPCR, when sufficiently well optimised, has enormous potential for the accurate quantification of viral RNA. However, significant bias can be observed when various target sequences and primer and probe chemistries are applied, reinforcing the importance of careful evaluation of the RT- dPCR method performance and quality control measures.