Arabidopsis thaliana to facilitate gene expression studies in this widely used plant model. We present optimised manual and automated protocols for the extraction of total RNA from 9-day-old Arabidopsis seedlings in a 96 well plate format using silica membrane-based methodology. Consistent and reproducible yields of high quality RNA are isolated averaging 8.9 μg total RNA per sample (~20 mg plant tissue). The purified RNA is suitable for subsequent qPCR analysis of the expression of over 500 genes in triplicate from each sample. Using the automated procedure, 192 samples (2 × 96 well plates) can easily be fully processed  in less than half a day. Additionally we demonstrate that plant samples can be stored in RNAlater at -20°C (but not 4°C) for 10 months prior to extraction with no significant effect on RNA yield or quality. Additionally, disrupted samples can be stored in the lysis buffer at -20°C for at least 6 months prior to completion of the extraction procedure providing a flexible sampling and storage scheme to facilitate complex time series experiments.The increasing popularity of systems-based approaches to plant research has resulted in a demand for high throughput (HTP) methods to be developed. RNA extraction from multiple samples in an experiment is a significant bottleneck in performing systems-level genomic studies. Therefore we have established a high throughput method of RNA extraction from  Systems biology involves the study of molecules in context as part of a larger system or network rather than in isolation and the development of mathematical models of the particular system being studied. Both the wet (laboratory-based) and dry (computer-based) experiments are used to inform each other and together generate a greater understanding of the biological system ,2. Over Arabidopsis thaliana seedlings. Depending on the available equipment, the method can be performed manually using multichannel pipettes or automated with a liquid handling robot. The developed protocols enable the study of gene expression by qPCR  in large numbers of samples .Historically, RNA extraction has been performed using organic solvents and phenol-chloroform . Similarlater solution .• RNA• 1.5 ml eppendorf tubes.http://www.mbpinc.com/, Cat.# 3453). NOTE: If using alternative tubes, ensure that they are sufficiently strong to withstand bead beating in a mixer mill.• 2 ml eppendorf tubes .• 5 mm stainless steel cone balls  which in addition to the binding, wash and elution plates, contains buffers RA1, RA2, RA3 & RA4 and DNaseI. The kit requires the addition of 1% β-mercaptoethanol to buffer RA1 and ethanol to buffers RA3 and RA4 prior to use.• illustra RNAspin 96 RNA isolation kit .http://www.starlab.co.uk/, Cat.# E2896-0110).• 1.1 ml 96 deepwell plates . To process 1× 96 well plate requires 8× 200 μl tips  and 144× 1000 μl tips .• Disposable tips for robot .http://www.binder-world.com).• Oven for preparing beads .• Bead-mill .• Plate centrifuge at room temperature capable of speed of 3,220× g (e.g. Eppendorf 5810R centrifuge fitted with an A-4-81 swing-bucket rotor).http://www.nanodrop.com).• NanoDrop spectrophotometer Agilent 2100 Bioanalyser  with 8-channel liquid handling arm fitted with disposable tips (200 μl and 1 ml as detailed above) and a TeVacS vacuum unit. Although we used the vacuum unit with a custom built spacer to ensure the wash plate  was positioned at the correct height directly below the binding plate, a standard size spacer (number 6) can be used. Details of the robot deck layout are depicted in Figure Although our method was developed and performed on a high specification liquid handling robot, it can be easily transferred to other platforms.• Freedom Evo-2 150 liquid handling robot  seedlings carrying the ELF3:LUC transgene (EARLY FLOWERING 3) [CIRCADIAN CLOCK ASSOCIATED 1) seedlings [CAB2:LUC transgene (CHLOROPHYLL A/B-BINDING PROTEIN 2) [-2·s-1) at 22°C for 9 days.Wildtype ERING 3)  or CCA1-eedlings  carryingOTEIN 2)  were groThe concentration of purified RNA was measured using a NanoDrop spectrophotometer (ND1000). Typically 2 μl of each sample was used.http://www.agilent.com/) according to the manufacturer's instructions. The ladder used was RNA 6000 ladder  and 150-200 ng of each sample was loaded. All samples and the ladder were denatured at 70°C for 2 mins prior to analysis. The results were analysed using Agilent 2100 Expert software.The integrity and quality of purified RNA were analysed using an Agilent 2100 Bioanalyzer and a RNA 6000 Nano Assay kit  according to the manufacturer's instructions. cDNA was diluted 1/10 and 1 μl used for subsequent qPCR. qPCR analysis for isopentenyl pyrophosphate:dimethylallyl pyrophosphate isomerase 2 (IPP2) in a final volume of 10 μl was then performed using LightCycler 480 SYBR Green I Master mix on a LightCycler480 . The following primers each at 300 nM were used: IPP2 forward primer GTATGAGTTGCTTCTCCAGCAAAG and IPP2 reverse primer GAGGATGGCTGCAACAAGTGT [Purified total RNA (1 μg) was reverse transcribed into cDNA using SuperScript VILO cDNA synthesis kit with oligo dT primers . Only half of the lysed sample was processed further to complete the RNA extraction procedure while the other half was stored in the freezer (-20°C) after lysis as backup in the case of some experimental catastrophe.later.1) Using clean tweezers, harvest 45-50 mg Arabidopsis seedlings per sample directly into 1.5 ml eppendorf tubes containing 500 μl RNAlater solution.2) Store samples overnight at 4°C to allow full penetration of the RNA: At this point, samples can safely be stored at -20°C for up to 10 months with no detrimental effect - see comments section below.3) Store samples at -20°C until sample lysis and completion of the RNA extraction procedure. NOTEIf the balls are not rinsed properly the acid not only causes rust and corrosion damage to the balls but would also degrade the RNA sample.4) Before use, 5 mm stainless steel cone balls (one per sample) are prepared by washing in 0.4 M HCl for 1 h, thoroughly rinsed using RNase-free water and then baked at 240°C for at least 4 h. NOTE: 5) Add prepared cone balls to 350 μl RA1 (containing 1% β-mercaptoethanol) in a 2 ml eppendorf tube (one per sample per tube).later solution and dab dry on some tissue paper.6) Carefully remove seedlings from RNA7) Transfer seedlings to prepared eppendorf tube containing stainless steel cone ball and RA1 lysis buffer (containing 1% β-mercaptoethanol).-1 for 2 × 2 mins. The tube positions are rotated between shaking steps as described in the manufacturer's operating manual. NOTE: Although the TissueLyser equipment we used is capable of processing 192 samples (2 × 96) in 1.5 ml tubes simultaneously, the size of the bead  required for efficient sample disruption and homogenisation necessitated the use of 2 ml eppendorf tubes. Consequently, samples have to be processed in two batches of 48. Although it is possible to use smaller stainless steel balls (e.g. 3-4 mm) and thus process samples in larger batches of 96 × 1.5 ml eppendorf tubes, we have found that this only works well with wild type samples or similar. In our hands, smaller bead sizes do not work well with mutant plant samples which often grow poorly and the resulting yield is very low. As our experiments usually include a variety of genetic manipulations, we routinely use 5 mm cone balls for efficient disruption and homogenisation of all samples.8) Homogenise the samples in two batches of 2× 24 tubes using a TissueLyser bead mill at a frequency of 25 s9) Centrifuge lysed samples at 11,000 × g for 5 mins.10) Transfer supernatant to a filter plate.: Missing out this step subsequently results in clogged wells in the binding plate and drastically reduced yields.11) Centrifuge filter plate (over a 1.1 ml deepwell plate) at 3,200 × g for 10 mins to remove debris. NOTEAt this point, lysed samples can be safely stored at -20°C for up to 6 months with no detrimental effects on purified RNA - see comments section below.12) 150 μl of the cleared lysed solution is transferred to another 1.1 ml deepwell plate to be processed for RNA extraction while the remainder is stored at -20°C as backup in case of experimental disaster. NOTE: either a centrifuge or vacuum manifold to pass the solutions through the plate. NOTE: We have modified the kit manufacturer's protocol to be used on plate centrifuges commonly found in labs  and all centrifugation steps were carried out in an Eppendorf 5810R centrifuge with an A-4-81 swing-bucket rotor. The plate sandwich formed by the binding plate combined with the plate included in the kit was too deep to be used in our centrifuge set up. Therefore the plate sandwich was composed of a binding plate over a 1.1 ml 96 deepwell plate not the plate provided with the kit. All vacuum steps were performed at -200 mbar for 2 × 60 secs. In our hands, greater pressure resulted in splashing of the sample and possible cross well contamination.The manual RNA extraction procedure is performed using multichannel pipettes and 13) Prepare solutions as detailed in the illustra RNAspin96 kit instructions (making DNaseI solution and adding ethanol to RA3 and RA4 buffer concentrates).14) Prepare cleared lysed samples  for binding to the silica membrane by adding an equal volume of RA4 buffer (150 μl) to each well of 1.1 ml deepwell plate.15) Mix thoroughly by pipetting up and down 12 times.16) Transfer samples (300 μl) to the wells of the binding plate.17) Apply vacuum  OR centrifuge  to pull the solution through the binding plate.18) Add 460 μl buffer RA3 to each well of the binding plates .19) Apply vacuum  OR centrifuge  to pull the solution through the binding plate.20) Add 29 μl DNase I solution to the centre of each well of the binding plate and incubate for 10 mins.21) Add 460 μl RA2 buffer to each well of the binding plate.22) Apply vacuum  OR centrifuge  to pull the solution through the binding plate.23) Add 800 μl RA3 buffer to each well of the binding plate.24) Apply vacuum  OR centrifuge  to pull the solution through the binding plate.25) Add 460 μl RA4 buffer to each well of the binding plate.26) Apply vacuum  OR centrifuge  to pull the solution through the binding plate.27) Proceed to elution (step 30 below).The automated RNA extraction procedure is performed using a liquid handling robot with an integrated vacuum unit on the robot deck to pass the solutions through the binding plate.13) Prepare solutions as detailed in the illustra RNAspin96 kit instructions (making DNaseI solution and adding ethanol to RA3 and RA4 buffer concentrates).14) Prepare the robotic workstation and check that the actual deck layout matches the virtual one shown on the robot script with all labware  containing appropriate solutions  in the correct locations on the robot deck - see Figure 15) Place 1.1 ml deepwell plate containing 96 lysed samples  onto the appropriate position of the robot deck.16) Select appropriate robot script and run which will perform steps 17-28 as detailed below.17) Prepare lysed samples for binding to the silica membrane by the addition of an equal volume of RA4 buffer (150 μl) and mix thoroughly by pipetting (150 μl) up and down 12 times.18) Transfer samples (300 μl) to the wells of the binding plate.19) Apply vacuum  to pull the solution through the binding plate.20) Add 460 μl buffer RA3 to each well of the binding plates .21) Apply vacuum  to pull the solution through the binding plate.22) Add 29 μl DNase I solution to the centre of each well of the binding plate and incubate for 10 mins.23) Add 460 μl RA2 buffer to each well of the binding plate.24) Apply vacuum  to pull the solution through the binding plate.25) Add 800 μl RA3 buffer to each well of the binding plate.26) Apply vacuum  to pull the solution through the binding plate.27) Add 460 μl RA4 buffer to each well of the binding plate.28) Apply vacuum  to pull the solution through the binding plate.29) Remove binding plate from robot and proceed to elution (step 30 below).Regardless of whether the manual or automated method is used, the same elution procedure is followed.30) Centrifuge the plate at 3,220 × g for 10 mins to ensure all ethanol is removed prior to elution.31) Add 50 μl RNase-free water to each well of the binding plate and incubate for 3 mins.32) Place binding plate over the 96 well elution plate (contained in the illustra kit) and elute purified RNA by centrifugation at 3,220 × g for 3 mins.CAB2:LUC transgene which are elongated [ELF3:LUC transgene.The yields of extracted RNA purified using the manual methods (using a centrifuge or vacuum) and the automated (using vacuum) procedure were similar Figure . Althouglongated . As the longated , these 9260/280 ratio of between 1.8 and 2.2 (average 2.09) indicative of highly pure RNA [260/230 ratios, which are a measure of polysaccharide contamination, were much more variable. This often occurs with plant samples which typically have relatively high polysaccharide contents [260/230 ratio seemed to have no detrimental effect on subsequent use of the purified RNA for reverse transcription and real time-qPCR (see comments below). This may not be the case for other applications which would have to be individually assessed.The quality of the HTP purified RNA was assessed by UV spectrometry using a Nanodrop spectrophotometer Figure . All purpure RNA . The A26contents -18 howevThe integrity and quality of purified RNA can be analysed using denaturing gel electrophoresis and visualising the ribosomal RNA bands. However, this technique requires a relatively large amount of RNA to detect clearly visible bands and the RNA is not recoverable for subsequent use. Recently, this analysis has been largely replaced by the use of Bioanalyzers which use microfluidics and capilliary electrophoresis on chips. Bioanalyzers require much smaller amounts of RNA and are ideal when limited amounts of starting sample are available. We therefore assessed the purified RNA using Bioanalyzer microfluidic chips which demonstrated its consistently high quality producing distinct, sharp ribosomal peaks Figure .isopentenyl pyrophosphate:dimethylallyl pyrophosphate isomerase 2 (IPP2) gene expression was performed on eight scattered samples  from one of the 96 well plates  is sufficient for subsequent cDNA synthesis and analysis of expression by qPCR of over 500 genes in triplicate (based on single-plex qPCR) using our usual procedures [[Plant tissue contains large amounts of polysaccharides and a number of endogenous PCR inhibitors such as polyphenolic compounds which can co-purify with the RNA and inhibit downstream applications such as qPCR -18. The bidopsis . All tescedures [ and unpuTraditional methods of RNA extraction such as phenol-chloroform are cheaper to perform and produce a greater yield than kit based silica methods . However, modern health and safety practice encourages the use of safer alternatives (where available) to replace harmful or dangerous chemicals so, even on low throughput, many labs now routinely use silica-based methods (such as spin columns). In addition to simplifying sample handling procedures, the use of a 96 well plate format is cost effective as the cost per sample in the 96 well format is less than half that incurred when using individual mini spin columns. This is before other considerations such as staff time are factored in and represents a considerable saving for larger scale studies involving hundreds of samples. The 96 well plate format also lends itself to automation. Liquid handling robots used to be the preserve of industry however they are becoming more commonplace in academia particularly with the recent move to a more systems-based approach to studying biology. Liquid handling robots are also decreasing in price and becoming more affordable both for core facilities and individual laboratories. Small systems cost around the same as a plate format qPCR machine and can be used for multiple applications as many individual labs will not have sufficient sample throughput for a dedicated robot specifically for RNA extraction. Our optimised protocol can easily be implemented on other robot platforms providing flexibility to the available system.The automated RNA purification procedure is based on the adsorption of nucleic acids to silica membranes and can be performed on the open bench in contrast to a recently published HTP method using phenol chloroform  which relater used in our protocol) can be used. These have recently become commercially available and increasingly popular  or 10 months . Storage at 4°C also affects the quality of the RNA.Many of the issues encountered with plant RNA extraction protocols are thought to originate from the initial sampling procedure . In orde example ). These C Figure . AlthougWe also investigated the effects of freezing the lysed samples in RA1, the lysis buffer supplied with the illustra kit. Samples were processed immediately or after storage at -20°C for 2 days or 6 months prior to completion of the extraction procedure Figure . No signlater) and the ability to freeze lysed samples with no detrimental affects on the subsequently isolated RNA, provide a flexible sampling and storage scheme. When this is used in conjunction with our automated HTP RNA extraction procedure, it enables complex time series experiments critical for systems-based plant research.We describe a HTP silica membrane-based method for total RNA extraction from Arabidopsis seedlings in a 96 plate format. This procedure can be performed manually by using appropriate multichannel pipettes or automated with the use of a specialised liquid handling robot. The described protocols isolate RNA in sufficient quantity and of high quality suitable for sensitive downstream processes such as reverse transcription and qPCR. The procedure is time efficient and cost effective for the processing of large numbers of samples. We have used our automated method to investigate and verify sample storage options during the procedure. The combined use of an aqueous RNA stabilising solution (RNAThe authors declare that they have no competing interests.ESC and SK performed the experimental work and performed the data analysis. LEK conceived and coordinated the project and wrote the manuscript. All authors have read and approved the final manuscript.


IRF1. RNAi-mediated depletion of the ubiquitin hydrolase, USP22 increases ubH2B levels and decreases transcriptional elongation at IRF1. Unexpectedly, 3’-end cleavage and polyadenylation of IRF1 is diminished, leading to a 2-to-3-fold increase in unprocessed IRF1 transcripts. The polyadenylation factor, CPSF73 is not effectively recruited and serine 2 phosphorylation (Ser2P) of the C-terminal domain of RNA polymerase II is disrupted. Two other JAK-STAT inducible transcripts are similarly affected, while two constitutively expressed transcripts are not. A working model, wherein a cycle of H2B ubiquitination/deubiquitination specifies Ser2P to regulate elongation and 3’-end processing of JAK-STAT inducible mRNAs is proposed. These results further elaborate USP22 function and its role as a putative cancer stem cell marker.5’-capping, splicing and 3’-cleavage/polyadenylation of a nascent RNA transcript are all coupled to transcription. Transcription is regulated by the post-translational modification of histones at the chromatin template. How histone modifications affect cotranscriptional RNA processing, however, is not well understood. By studying how the chromatin template contributes to the dynamic gene expression induced downstream of the JAK-STAT signaling pathway, we have uncovered relationships between specific histone modifications and RNA processing. Here we report that H2B monoubiquitination (ubH2B) is required for the effective 3’-end processing of the JAK-STAT inducible transcript,


Rheum australe) and arnebia (Arnebia euchroma). A rapid and less cumbersome system for isolation of RNA was essential to facilitate any study related to gene expression.Secondary metabolites are reported to interfere with the isolation of RNA particularly with the recipes that use guanidinium-based salt. Such interference was observed in isolation of RNA with medicinal plants rheum , differential display (DD), suppression subtractive hybridization (SSH) library construction, and northern hybridization. Litchi chinensis, Pinus taeda, Pseudotsuga menziesii, Picea glauca, Griffonia simplicifolia and Albizia procera , sodium acetate  and ethylenediaminetetra acetic acid .• Chloroform• 70% Ethanol• Isopropanoli. Grind 10-100 mg of tissue to a fine powder in liquid nitrogen using a mortar and pestle.ii. Add 2 ml of solution I, grind further. Solution I gets frozen as added; make fine powder of the frozen material; continue grinding so as to make a homogenous mixture; this ensures close contact of the tissue ingredients and the reagents that would help in instantaneous denaturation of protein. Allow to thaw completely with intermittent grinding.iii. Add 800 μl of DEPC-treated water, and mix it by grinding.iv. Transfer the contents to two, 2 ml micro-centrifuge tubes and leave for 5 min at room temperature.v. Add 200 μl of chloroform to each tube, vortex briefly (< 10 s) and leave for 10 min at room temperature.vi. Centrifuge at 13,000 rpm for 10 min at 4 °C and transfer the upper aqueous phase into fresh tubes.vii. Add 0.6 volumes of isopropanol, vortex briefly (< 10 s) and leave for 10 min at room temperature.viii. Centrifuge at 13,000 rpm for 10 min at 4 °C and discard the supernatant.ix. Wash RNA pellet with 70% ethanol, air dry and dissolve in 20 to 50 μl of DEPC-treated water.26 S rRNA [Purity and concentration of RNA was assessed by determining the absorbance of the sample at 260 and 280 nm using a spectrophotometer . Integrity of RNA was evaluated on a denaturing formaldehyde-agarose gel as described previously . First s6 S rRNA . Isolate6 S rRNA ,15-17.A phenol-based, guanidinium salt-free protocol for isolation of RNA was developed. Reagents were selected based on their specific properties: phenol as a strong protein denaturant and inhibitor of RNase; SDS and EDTA are also inhibitors of RNase . This coThe developed method successfully isolated RNA from leaf tissues of rheum and arnebia with a yield of 168.66 ± 4.7 and 153.33 ± 11 μg RNA/100 mg of tissue, respectively. This was remarkable in the light that other protocols either failed to yield RNA or yielded extremely low quantity Figure . In factA260/280 ratio of the isolated RNA ranged between 1.8 - 2.0, which indicated the RNA to be relatively free of proteins and contaminants. At times, low A260/280 ratio ranging between 1.4 - 1.7 was also obtained, particularly when higher amount of the tissue was used. In all the cases, distinct 28 S and 18 S rRNA bands were observed on 1% formaldehyde-agarose denaturing gel indicating intact RNA. Also, the isolated RNA was amenable to downstream applications e.g. RT-PCR, DD, northern analysis, SSH and RACE, as reported in our publications that utilized the present protocol [The method so developed was further extended to nineteen more tissues Figure  from difprotocol -17,22,23Unlike specific protocols for different tissues, the developed method was very useful in isolation of RNA not only from rheum and arnebia but also from various other plant species belonging to diverse genera. The distinguishing feature of the protocol was the success with those tissues wherein the commonly used protocols failed. The protocol is simple, does not require any specialized material, chemical, instrument and procedure such as ultracentrifugation step through cesium chloride gradient or lithium chloride precipitation, thereby greatly reducing the complications and the time required.The authors declare that they have no competing interests.SG standardized various solutions and optimized protocol. AP, HK, AK, HS, PKB, AR, RSS, JR and KS carried out the experiments and participated in manuscript writing along with SG. SK guided on development of the RNA isolation system, guided the research and finally drafted the manuscript. All the authors have read the manuscript and agree with the content.


Pospiviroidae) and the chloroplast (family Avsunviroidae). Viroid replication proceeds through an RNA-based rolling-circle mechanism with three steps that, with some variations, operate in both polarity strands: i) synthesis of longer-than-unit strands catalyzed by either the nuclear RNA polymerase II or a nuclear-encoded chloroplastic RNA polymerase, in both instances redirected to transcribe RNA templates, ii) cleavage to unit-length, which in the family Avsunviroidae is mediated by hammerhead ribozymes embedded in both polarity strands, while in the family Pospiviroidae the oligomeric RNAs provide the proper conformation but not the catalytic activity, and iii) circularization. The host RNA polymerases, most likely assisted by additional host proteins, start transcription from specific sites, thus implying the existence of viroid promoters. Cleavage and ligation in the family Pospiviroidae is probably catalyzed by an RNase III-like enzyme and an RNA ligase able to circularize the resulting 5′ and 3′ termini. Whether a chloroplastic RNA ligase mediates circularization in the family Avsunviroidae, or this reaction is autocatalytic, remains an open issue.Viroids, due to their small size and lack of protein-coding capacity, must rely essentially on their hosts for replication. Intriguingly, viroids have evolved the ability to replicate in two cellular organella, the nucleus (family  This approach, combined with RNase protection assays, has identified the initiation sites of ASBVd ml (+) and (−) RNAs isolated from infected avocado at similar (A+U)-rich terminal loops in their predicted quasi-rod-like secondary structures [ml (+) and (−) RNAs isolated from infected peach at similar double-stranded motifs of 6 to 7 bp that also contain the conserved GUC triplet preceding the self-cleavage site in both polarity strands; within their predicted branched secondary structures, the motifs are located at the base of similar long hairpins that presumably contain the NEP promoters [in vitro transcriptions with truncated PLMVd RNAs and the RNA polymerase of E. coli [in vitro a bona fide initiation complex reproducing the in vivo situation, particularly with a eubacterial RNA polymerase very different from NEP. Therefore, the structural motifs containing the transcription initiation sites are clearly distinct in the two viroids: terminal loops in ASBVd and double-stranded motifs in PLMVd.The question of the transcription initiation sites has been undertaken by ructures . The samromoters . These Promoters , are dif E. coli . This di4.2.Avsunviroidae [in vivo of the multimeric viroid replicative intermediates wherein they are embedded, have been reviewed elsewhere [Apart from the viroid discovery itself, the finding —first in ASBVd and then in the other members of the family viroidae ,50— thatviroidae . Previoulsewhere ,50. Heretrans  showed that efficient cleavage in vitro requires Mg2+ concentrations of 5–10 mM, an order of magnitude higher than that existing in vivo. However, re-examinations in vitro and in vivo have demonstrated that natural cis-acting hammerheads with intact loops 1 and 2 self-cleave much faster (retaining activity at 0.5-1 mM Mg2+), and that modifications of loops 1 and 2 cause a severe reduction of their catalytic activity [in vitro and for viroid infectivity in vivo, whereas loop 2 has a key opened A at its 3′-side. These structural features promote a specific loop-loop interaction motif across the major groove, the structural features of which —base pairing between the 5′ pyrimidine of loop 1 and the 3′ purine of loop 2, and interaction of the extrahelical pyrimidine of loop 1 with loop 2  with loose sequence requirements and usually capped by short loops . X-ray crystallography has unveiled that the actual conformation of these catalytic motifs does not resemble a hammerhead, as suggested by the initial bidimensional representation, but rather a Y wherein stems III and II are almost colinear . Data obactivity ,59, thusactivity ,61. Moreh loop 2 — are likin vivo by chloroplastic proteins acting as RNA chaperones [Avsunviroidae may be tackled with a system based in transplastomic Chlamydomonas reinhardtii expressing (+) and (−) dimeric transcripts; despite the absence of viroid RNA-RNA transcription, this system can be used to identify the cellular factors facilitating cleavage  [Two additional points regarding hammerhead function in the natural context deserve a comment. First, the loop-loop tertiary interactions could be further stabilized aperones . Furthere below) . And sece below) . Therefo4.3.A. thaliana is the only plant RNA ligase well-characterized biochemically and genetically [A. thaliana and Oryza sativa two of the splicing enzymes, the tRNA ligase and 2′-phosphotransferase , have transit signals at their N-termini and are predominantly targeted to chloroplasts and proplastids, respectively [Avsunviroidae. Moreover, in one viroid-like satellite RNA the junction resulting from ligation of the two termini generated by hammerhead-mediated self-cleavage contains the expected signature of a tRNA ligase: a 2′-phosphomonoester, 3′, 5′-phosphodiester group [in vitro self-cleavage and infectivity. We suspect that this deletion may have been introduced during reverse transcription, because it was detected only in the plus-polarity strand, and that its ultimate cause could be the presence in a fraction of the viroid population of a 2′-phosphomonoester at the nucleotide preceding the self-cleavage/ligation site, which would lead to a jumping of the reverse transcriptase [in vitro self-ligation of PLMVd, assuming that this atypical bond is indeed present in vivo (see below).First, considering that the 5′-OH and 2′, 3′-cyclic phosphodiester termini resulting from hammerhead-mediated self-cleavage are those typically required by the wheat germ tRNA ligase, whose homologue from etically , and conetically . This loetically . Most imectively . Therefoer group . Encapsicriptase –73. AnotSecond, the hammerhead ribozyme may mediate not only self-cleavage but also ligation generating a 3′, 5′-phosphodiester linkage. This is indeed the case for another small ribozyme, the hairpin ribozyme, which is found embedded in certain viroid-like satellite RNAs . When coin vitro mostly producing 2′, 5′-instead of the conventional 3′, 5′-phosphodiester bonds [in vitro self-cleavage [in vitro self-ligation of PLMVd demands concentrations of Mg+2 around 100 mM, which are much higher than those existing in vivo, ii) in vitro self-ligation has also been observed in PSTVd [in vivo [in vitro [in vivo, while we have not observed prominent stops at the ligation site in our primer extension experiments using ASBVd circular forms isolated from infected tissue [Third, the reaction could also be autocatalytic (self-ligation) but operating through a different pathway. This hypothesis is based on the observation that the monomeric linear PLMVd RNAs resulting from hammerhead-mediated self-cleavage can self-ligate er bonds , and on cleavage . Howeverin PSTVd  and in Hin PSTVd , while n[in vivo ,32,80, ain vitro  and presd tissue .5.Pospiviroidae and of the RNA ligases catalyzing circularization in both families (if this step is indeed an enzyme-mediated reaction in the family Avsunviroidae).The lack of protein-coding capacity of viroids entails that their replication mechanism is much more host-reliant than that of RNA viruses, which at least encode a subunit of the RNA-dependent RNA polymerase catalyzing initiation and elongation of viral strands. Moreover, viroids are able to redirect certain nuclear and chloroplastic RNA polymerases to transcribe RNA instead of their physiological DNA templates. Although the nature of the RNA polymerases involved in viroid replication appears well-established, they most likely act in concert with other host proteins that facilitate their template switching and processivity. New research avenues include identification of these proteins, as well as of the RNase mediating cleavage of oligomeric (+) replicative intermediates in the family Avsunviroidae —essentially limited to the natural hosts in which they were first reported [On the other hand, viroid replication may be closely linked to other biological features. Here are three illustrative examples. First, is viroid pathogenesis dependent or independent of replication? Although there is a report indicating that the ectopic expression of non-replicating viroid sequences incites typical symptoms , a recenreported ,49, is ireported , is it a


Saccharomyces cerevisiae and fission yeast Schizosaccharomyces pombe, the lncRNA-based chromatin regulations of germ cell differentiation are quite different. In this review, we present an overview of these regulations by focusing on the mechanisms and their respective functions both in S. cerevisiae and in S. pombe. Part of these lncRNA-based chromatin regulations may be conserved in other eukaryotes and play critical roles either in the context of germ cell differentiation or, more generally, in the development of multicellular organisms.Germ cell differentiation, the cellular process by which a diploid progenitor cell produces by meiotic divisions haploid cells, is conserved from the unicellular yeasts to mammals. Over the recent years, yeast germ cell differentiation process has proven to be a powerful biological system to identify and study several long noncoding RNAs (lncRNAs) that play a central role in regulating cellular differentiation by acting directly on chromatin. Remarkably, in the well-studied budding yeast  Schizosaccharomyces pombe (Lejeune and Allshire The widespread implication of nuclear long noncoding RNAs (lncRNAs) in the regulation of gene expression is now established in a broad range of eukaryotes  gene. IME1 gene encodes the master transcription regulator of sporulation, and ectopic expression of IME1 in diploid cells is sufficient to induce sporulation   Fig. . Thus, uIME1 promoter and it efficiently inhibits its transcription , tightly correlates to the growth and differentiation status of the yeast. In cells undergoing vegetative growth, IRT1 RNA level is relatively high, while upon and during sporulation it goes down , which, like IRT1, acts in cis   and M (or h−) conjugate to form a diploid cell competent for sporulation  res Fig. . At the mei3  RNA-binding protein family, which has members present in the vast majority of eukaryotes (Stoilov et al. mei4 gene (Harigaya et al. mei4 rescues most of the dramatic growth defect observed in mmi1∆ cells (Harigaya et al. mei4 mimics the meiotic block observed in sme2∆ cells (Kakui et al. sme2∆ meiotic block is mostly caused by a lack of Mei4 protein due to the constitutive activation of Mmi1 silencing machinery. Interestingly, the meiRNA species itself is a target of Mmi1-directed degradation during vegetative growth (Fig. The silencing imposed by Mmi1 RNA surveillance machinery is essential for proper vegetative growth (Harigaya et al. wth Fig. . This powth Fig. .sme2 locus exerts another remarkable function linked to chromatin. At the onset of meiosis I, the two sme2 loci strongly pair regardless of their chromosomal locations (Fig. sme2 pairing requires production of its long noncoding transcripts, the meiRNAs, but neither Mei2 nor Mmi1-mediated RNA degradation. This represents the first example of a RNA-mediated chromosome pairing. However, its potential conservation in other eukaryotes and the biological significance of this pairing remain to be addressed.In addition to its major role in neutralizing Mmi1-directed gene silencing, ons Fig. . This smS. pombe, formation of constitutive heterochromatin, characterized by histone H3K9 methylation and the presence of HP1-like proteins, takes place at relatively large, noncoding and repeated DNA found mostly at the pericentromeric, subtelomeric, and mating type regions (Grewal In s Grewal . In paras Grewal . The mecs Grewal  (Hiriarts Grewal . These fs Grewal . In paras Grewal  (Hiriarts Grewal . Howevers Grewal . Nonethes Grewal . The rolS. cerevisiae and S. pombe have diverged from a common ancestor at least 300 million years ago (Sipiczki S. cerevisiae, one mechanism imposes gene silencing by producing a lncRNA, within the promoter of a key gene for the induction of sporulation, to serve as a recruiting platform for histone modifying proteins that are believed to establish a repressive chromatin structure. In parallel, production of a noncoding antisense RNA, which may not have any function per se, contributes to silence another gene important for sporulation induction. In S. pombe, the long noncoding meiRNA species act together with Mei2 RNA-binding protein as a molecular pump or decoy to sequester Mmi1 RNA-binding protein, a key repressor of meiosis, within a specific region of the nucleus. In addition, Mmi1 imposes a potent gene silencing of meiotic genes in vegetative cells, by triggering selective RNA degradation, and inhibiting splicing. Mmi1, by binding to nascent transcripts of protein-coding genes, also induces the formation of facultative heterochromatin that may participate to the silencing of its target genes. Altogether, the variety of RNA-dependent mechanisms implicated in the regulation of yeast germ cell differentiation illustrates the versatility of processes that can be used by RNA to control a key cell-fate decision. An important open question is whether such mechanisms are conserved in other eukaryotes including mammals and whether they exert similar functions.It is estimated that Sipiczki . Thus, tstra8 expression is controlled by the RNA-binding protein Nanos2 (Suzuki and Saga In animals, the entry of germ cell differentiation has been quite extensively studied, notably in mouse Saga . The mouS. pombe remains to be determined. Ongoing studies on yeast lncRNA-based chromatin regulation in the context of germ cell differentiation should continue to provide important insights into our understanding of how lncRNAs regulate germ cell differentiation and development from yeasts to mammals.In animal germ cell differentiation, RNA-based gene silencing exerts an essential function towards transposon. Part of this mechanism is believed to go through long and small RNA-mediated chromatin silencing (Bourc’his and Voinnet


Recent advances in DNA sequencing have made it possible to sequence the whole transcriptome by massively parallel sequencing, commonly referred as RNA-Seq. RNA-Seq is quickly becoming the technology of choice for transcriptome research and analyses. RNA-Seq allows to reduce the sequencing cost and significantly increase data throughput, but it is computationally challenging to use such RNA-Seq data for reconstructing of full length transcripts and accurately estimate their abundances across all cell types. A number of recent works have addressed the problem of transcriptome reconstruction from RNA-Seq reads. These methods fall into three categories: genome-guided, genome-independent and annotation-guided.Transcriptome Reconstruction using Integer Programing” (TRIP) that incorporates fragment length distribution into novel transcript reconstruction from paired-end RNA-Seq reads. To reconstruct novel transcripts, we create a splice graph based on inferred exon boundaries and RNA-Seq reads. A splice graph is a directed acyclic graph (DAG), whose vertices represent exons and edges represent splicing events. We enumerate all maximal paths in the splice graph using a depth-first-search (DFS) algorithm. These paths correspond to putative transcripts and are the input for the TRIP algorithm.In this work, we propose a novel statistical genome-guided method called “To solve the transcriptome reconstruction problem we must select a set of putative transcripts with the highest support from the RNA-Seq reads. We formulate this problem as an integer program. The objective to select the smallest set of putative transcripts that yields a good statistical fit between the fragment length distribution empirically determined during library preparation and fragment lengths implied by mapping read pairs to selected transcripts.Preliminary experimental results on synthetic datasets generated with various sequencing parameters and distribution assumptions show that TRIP has increased transcriptome reconstruction accuracy compared to previous methods that ignore fragment length distribution information.


BUD27 affects growth and leads to a substantial accumulation of the three RNA polymerases in the cytoplasm, defects offset by the overexpression of RPB5. Supporting this, our data demonstrate that the lack of Bud27 affects the correct assembly of Rpb5 and Rpb6 to the three RNA polymerases, suggesting that this process occurs in the cytoplasm and is a required step prior to nuclear import. Also, our data support the view that Rpb5 and Rpb6 assemble somewhat later than the rest of the complexes. Furthermore, Bud27 Rpb5-binding but not PFD-binding domain is necessary for RNA polymerases biogenesis. In agreement, we also demonstrate genetic interactions between BUD27, RPB5, and RPB6. Bud27 shuttles between the nucleus and the cytoplasm in an Xpo1-independent manner, and also independently of microtubule polarization and possibly independently of its association with the RNA pols. Our data also suggest that the role of Bud27 in RNA pols biogenesis is independent of the chaperone prefoldin (PFD) complex and of Iwr1. Finally, the role of URI seems to be conserved in humans, suggesting conserved mechanisms in RNA pols biogenesis.The unconventional prefoldin URI/RMP, in humans, and its orthologue in yeast, Bud27, have been proposed to participate in the biogenesis of the RNA polymerases. However, this role of Bud27 has not been confirmed and is poorly elucidated. Our data help clarify the mechanisms governing biogenesis of the three eukaryotic RNA pols. We show evidence that Bud27 is the first example of a protein that participates in the biogenesis of the three eukaryotic RNA polymerases and the first example of a protein modulating their assembly instead of their nuclear transport. In addition we demonstrate that the role of Bud27 in RNA pols biogenesis depends on Rpb5. In fact, lack of  Saccharomyces cerevisiae mediates the correct assembly of the three complexes prior to their translocation to the nucleus, in a process which is dependent on Rpb5. In addition, our data support the view that, during the assembly of the RNA pols, Rpb5 and Rpb6 assemble rather late compared to the rest of the complexes. Furthermore, this role of Bud27 seems to be specific, as it is not extended to other prefoldin members. Finally, the role of Bud27 seems to be conserved in humans, suggesting conserved mechanisms in RNA pols biogenesis.The mechanisms governing the assembly and the transport of the three eukaryotic RNA polymerases to the nucleus are in discussion. Interesting papers have demonstrated the participation of some proteins in the assembly of the nuclear RNA polymerases and in their transport to the nucleus, but the mechanisms involved are poorly understood. Our data help clarify the mechanisms governing biogenesis of the three eukaryotic RNA pols and demonstrate that the prefoldin Bud27 of  Eukaryotic RNA polymerases are a family of multimeric enzymes, RNA pol I, II, and III, responsible for the specific synthesis of different RNAs. RNA pol I is specialized in the synthesis of the pre-rRNA precursor of the three largest rRNA and typically account for about 75% of the entire transcription output in fast-growing yeast cells. RNA pol III transcribes mostly tRNAs and 5S rRNA, together with several short non-translated RNAs, while transcription corresponds to about 15% of the total RNA. RNA pol II, the enzyme that produces all mRNAs and many non-coding ones, transcribes most of the nuclear genome but nevertheless contributes to less than 10% of total RNA in growing cells. RNA pol I, II, and III are composed of 14, 12, and 17 subunits respectively, with a catalytic core formed by the two largest subunits highly conserved through evolution and five common subunits to the three enzymes Noteworthy findings in both human and yeast demonstrate the participation of different proteins in the transport of the RNA pol II to the nucleus, Iwr1 and Npa3 in yeast, and GPN1 (RPAP4) and GPN3 in humans Saccharomyces cerevisiae, only a cytoplasmic localization has been detected In humans, R2TP/Prefoldin-like complex contains Rpb5, a common subunit to the three eukaryotic RNA polymerases BUD27 leads to a substantial accumulation of the three RNA polymerases in the cytoplasm, a defect offset by the overexpression of RPB5. Supporting this, our data demonstrate that the lack of Bud27 affects the correct assembly of Rpb5 and Rpb6 to the three RNA polymerases, suggesting that this process occurs in the cytoplasm and is a required step prior to nuclear import. As previously proposed in human BUD27, RPB5 and RPB6. In addition, Bud27 PFD- or Rpb5-binding domains are not necessary for RNA polymerases biogenesis. Finally, our data demonstrate that Bud27 shuttles between the cytoplasm and nucleus, as shown by the deletion of the NES domain, contrary to what has been previously described, a mechanism that is independent of the Xpo1-mediated pathway. URI silencing suggests that similar role for this protein accounts in humans.Despite that some experiments, using immunoprecipitation and mass-spectrometry, have shown that URI interacts with RNA polymerases and other intermediary subcomplexes involved in RNA pol II cytoplasmic assembly, very little is known about the role of URI in RNA polymerases biogenesis. In this report, we investigate the role of the URI yeast homologue Bud27 in RNA polymerases assembly and its relationship with Rpb5. Bud27 interacts with different subunits of the three RNA polymerases, with the common subunit Rpb5 and Rpb10, as well as with a component of the prefoldin-like complex, Yke2 (Pfd6). Deletion of BUD27 gene TAP-tagged at its 3′ end in TAP purifications. As shown, the Bud27-TAP cells grow normally  and Rpc25-Myc tagged (RNA pol III) proteins. As shown in DrosophilaBUD27-GFP gene fusion, cloned in a centromeric plasmid, expressed from a Tet-repressible promoter bud27 mutant strain  and Rpc160-Myc tagged (RNA pol III) proteins, using anti-HA, anti-Rpb1 (8WG16) and anti-Myc antibodies, to analyse the intracellular localization of the largest subunits of the RNA pol I, II, and III, respectively. In a wild-type strain . We performed immunoprecipitation experiments using anti-Rpb1 antibodies (8WG16) and analysed different RNA pol II subunits corresponding to the different assembly intermediate previously described, Rpb1 together with Rpb4/5/7/8/9 and Rpb2 together with Rpb3/10/11/12 To investigate the effect of Bud27 in RNA pols complex assembly, we immunoprecipitated RNA pol II from a wild-type and a Δbud27 mutant strain . The immunoprecipitation of the three largest RNA pol subunits, using anti-HA, anti-Rpb1 (8WG16), and anti-Myc antibodies, again revealed significant differences in yield between Rpb5 and the largest subunits of the three RNA pols between wild-type and mutant polymerase complexes , as Rpb3-TAP purification has been largely and successfully used to purify RNA pol II bud27 cells. It is also significant the reduction of Rpb3 co-purifying proteins. Taking together, our results suggest that lack of Bud27 led to an instable RNA pol II complex and then, that Bud27 is also necessary to maintain stability of the enzyme.To extend our analysis, we performed Rpb3-TAP purification from wild type and ΔThe data above strongly indicate that Bud27 is required for assembly of the three RNA pols and that this mechanism depends on correct assembly or stabilization of Rpb5. Because Rpb5 was identified as an interactor with Bud27, we considered the possibility that these proteins are functionally linked.RPB5 overexpression corrects the temperature sensitivity of the Δbud27 mutant strain. Notably, increasing dosage of Rpb5 suffices to rescue the growth defect of the strain lacking Bud27 at a restrictive temperature  under RPB5 overexpression. Immunoprecipitation of the three largest RNA pols subunits, using anti-HA, anti-Rpb1 (8WG16) and anti-Myc antibodies, revealed no significant differences in the yield between Rpb5 and the largest subunits of the three RNA pols between wild-type and mutant polymerase complexes  is believed to function as a scaffold protein able to assemble additional members of chaperone prefoldin (PFD) family through its PFD and Rpb5-binding domains in both human and yeast. Human and yeast PFD is a complex composed of six different subunits, PFD1-PFD6, referred to as the prefoldin/GimC complex, which functions as a molecular chaperone and delivers newly synthesised unfolded proteins to cytosolic chaperonin TRiC/CCT to facilitate the folding of proteins YKE2 (PFD6), shown by us and others GIM6 (PFD1), which genetically interacts with BUD27BUD27 nor with the RNA pols. Surprisingly, in contrast to Bud27, these three components of the prefoldin complex are dispensable for the nuclear localization of the three RNA pols at 30°C or even at 37°C  family of ATP-independent molecular chaperones, also called unconventional prefoldin Rpb5 interactor BUD27 and different components of the three transcriptional machineries have been found in yeast by us  and others Consistent with a role for Bud27 in RNA pols biogenesis, we demonstrated by TAP purification and immunoprecipitation experiments that Bud27 interacts with different subunits of the three RNA pols. Our data agrees with those of Krogan et al. showing physical interactions between Bud27 and RNA pol II BUD27 deletion accounts for a defect in nuclear transport. However, lack of Bud27 does not impair RNA pols nuclear localization under RPB5 overexpression, suggesting that Bud27 is dispensable for their nuclear import. Only two proteins in S. cerevisiae had been shown to participate in RNA pol II biogenesis so far, Iwr1 and Npa3, and only the Npa3 homologue RPAP4/GPN1 in humans. Notably, these proteins are necessary for nuclear transport but none of them are involved in pol assembly Biochemical and structural studies of RNA pols have proposed a detailed model of these enzymes, but however, little is known on how they assemble into the complexes or how they are transported from the cytoplasm to the nucleus. Our work provides new data to elucidate the mechanisms governing RNA pols biogenesis and localization and complement those in human and yeast bud27 mutant strain and the analysis of different subunits corresponding to the different assembly intermediate previously described RPB5 overexpression corrects not only assembly but also nuclear RNA pols transport, point to the fact that Bud27 mediates RNA pols assembly in an Rpb5-dependent manner, in agreement with data confirming its role in protein folding bud27 cells expressing a version of Bud27 lacking the Rpb5-binding domain show cytoplasmic RNA pols localization. In addition, the defect in Rpb6 assembly is also consistent with the fact that RPB6 overexpression partially corrects the temperature sensitivity of the Δbud27 mutant (our unpublished data) and with the physical contact between Rpb5 and Rpb6 on the RNA pol II structure BUD27 is not essential and its deletion seems only to affect part of the RNA pols complexes, it appears that Bud27 participates in coordination with other proteins to address its role in RNA pols biogenesis. These results also provide information concerning the mechanisms governing the assembly of Rpb5 and Rpb6 into the rest of the complexes. Our data suggest that Rpb6 could assemble rather late once Rpb5 is assembled, as previously proposed in human Our immunoprecipitation experiments in ΔAs is the case for Bud27 in yeast, HSP90 and its cochaperone RPAP3 in human cells have been shown to coordinate the assembly of RNA pol II and its involvement in RNA pol I and III assembly has been suggested DrosophilaAs shown here RNA pols nuclear localization is dependent on Rpb5-binding domain. However, Bud27 PFD and Rpb5-binding conserved domains are not required for growth, neither for its role in translation Finally, silencing experiments in human cells, account for a conserved role of URI in RNA pols biogenesis, suggesting similar mechanisms that must be deciphered.Common yeast media, growth conditions, and genetic techniques were used as described elsewhere Strains, plasmids and primers are listed in et al.Rpb8-ECFP and Rpb1-GFP tagging was performed by yeast recombination of a PCR fragment as described in Longtine 600∼0.6–0.8) in yeast extract-peptone-dextrose (YPD) medium or synthetic minimal (SD) were washed twice with ultrapure water and lysis buffer . Cells were resuspended in 1 ml lysis buffer supplemented with 1x protease inhibitor cocktail , 0.5 mM PMSF, 2 mM sodium orthovanadate and 1 mM sodium fluoride and whole-cell extracts were prepared using a MixerMill MM400 RETSCH (3 min 30 Hz). Immunoprecipitations were carried out as described elsewhere 400 ml cells growing exponentially (ATAP purification for protein identification by mass spectrometry was performed as described previously 600∼0.5–0.7), fixed with 37% w/v formaldehyde at room temperature for 2 h with slow shaking, and then centrifuged and washed twice with PBS. Cells were resuspended in spheroplasting buffer  and cell wall digested with 125 µg/ml zymolyase 20T  and 22.7 mM 2-mercaptoethanol (SIGMA) by incubation for 1 h at 37°C without shaking. The spheroplasts were washed twice with PBST (PBS with 0.05% Tween 20) and then resuspended in the same solution. Cell suspension was added to an AAS  slide, incubated at room temperature until slide was dry and washed twice with PBST. Then, 50 µl of PBS-BSA (1 mg/ml BSA) were added, and the slides. After incubation for 30 min in a humid chamber were washed three times with PBS. Next, 50 µl of 1∶100 dilution of the primary antibodies  in PBS-BSA (1 mg/ml BSA) were added and incubated 2 h at room temperature in a humid chamber. Slides were then washed three times with PBS, and incubated for 1 h, in the dark, at room temperature in a humid chamber with 50 µl of 1∶100 dilution of secondary antibody . The slides were washed three times with PBS and incubated for 5 min with 50 µl of 1 µg/ml DAPI (in PBS). After washing three times with PBS, slides were finally covered with a Vectashield (Vector Laboratories) mounting solution.Cells were grown at 30°C in YPD or SD medium (AHuman cells were fixed for 15 min at room temperature with 4% (v/v) paraformaldehyde in PBS. Following fixation, cells were washed three times for 15 min with PBS, treated with 50 mM ammonium chloride for 30 min and permeabilised for 20 min with PBS containing 0.1% (v/v) Triton X-100 (wash solution). Blocking solution (wash solution with 5% (w/v) BSA) was then added for 30 min and then, cells incubated for 1 h with 1∶100 dilution of the primary antibodies (8WG16) in blocking solution. Cells were washed three times for 15 min with wash solution and then incubated with 1∶100 dilution of secondary antibody  for 1 h in blocking solution. After washing three times with wash solution, 15 min each, slides were finally covered with a Vectashield (Vector Laboratories) mounting solution containing DAPI.The fluorescence intensity was scored with a fluorescence microscope (Olympus BX51).8 cells growing exponentially (A600∼0.6–0.8) were resuspended in 3 ml of 100 mM PIPES/KOH (pH 9.4) containing 10 mM DTT and 0.1% sodium azide and then incubated at room temperature for 10 min. Cells were spun down, resuspended in 2 ml of 50 mM phosphate buffer (pH 7.5), containing 0.6 M Sorbitol, 10 mM DTT, and 4 µl of 20 mg/ml zymoliase and incubated 10 min at 37°C in a water bath to spheroplast formation. Spheroplasts were then pelleted at 4°C, washed with 50 mM HEPES-HOK buffer (pH 7.5) containing 100 mM KCl, 2.5 mM MgCl2 and 0.4 M Sorbitol, resuspended in equal volume (∼80 µl) of EBX buffer (50 mM HEPES/KOH (pH 7.5), 100 mM KCl, 2.5 mM MgCl2, 0.25% Tritón-X100, 0.5 mM PMSF, 0.5 mM DTT, cocktail protease inhibitors Complete Roche 1x) and incubated for 3 min on ice. This whole cell extract was laid onto 400 µl of EBX-S buffer (EBX with sucrose 30%) and centrifuged at 12000 rpm for 10 min. After the sucrose gradient a chromatin pellet became visible and was washed with 400 µl of EBX buffer and finally resuspended in 100 µl of the same solution. A 1/10 dilution of chromatin pellet was used for SDS-PAGE and Western blotted with antibodies against Rpb1 (8WG16), alfa-tubulin  and Nop1 .Chromatin isolation was performed as previously described l-glutamine, and 50 U/ml penicillin–streptomycin. Cells were seeded in twenty four-well plates  and transfected in triplicate with 40–200 nM heteroduplex oligonucleotides using the Lipofectamine 2000 Transfection Reagent (Invitrogen), following the manufacturer's protocol, and incubated for 24 h at 37°C. Control cells were treated in the same conditions without siRNA heteroduplex. The experiments were performed three times (three replicates). After indicated time, cells were either harvested for RNA extraction or fixed with PFA 4% in PBS for inmunolocalisation analysis.URI, prefoldin-like chaperone (Gene ID: 8725) gene was silenced by transfection with the siRNA heteroduplex hsURI1_02 and hsURI1_02_as . Human pTotal RNA was isolated from URI silenced HPF cells using the SV Total RNA Isolation System (Promega), according to the manufacturers. Retrotranscription was performed from 200 ng of total RNA using the Maxima First Strand cDNA Synthesis Kit (Fermentas) in a final volume of 20 µl, according to the manufacturer's protocol. As a control, each sample was subjected to the same process without reverse transcriptase.URI mRNA accumulation was analyzed by q-RT-PCR with oligonucleotides hsURI1E9-f01 and hsURI1E10-r01 using cDNA corresponding to 10 ng. Human cyclophilin A (PPIA) was used as an internal control. Each PCR reaction was performed at least three times, with three independent samples. All oligonucleotides used are indicated in Figure S1bud27 mutant phenotypes are corrected by overexpression of different BUD27 constructions. Growth of wild-type and Δbud27 mutant strains transformed with different constructions containing whole BUD27 or deleted forms of BUD27, at different temperatures; pCM and pFL correspond to the control empty vectors.Δ(TIF)Click here for additional data file.Figure S2bud27 mutant cells overexpressing Bud27ΔNES-GFP were grown at 30°C and localisation of Bud27ΔNES-GFP and Rpb8-ECFP was analysed in vivo. B) BUD27 overexpression (pCM-BUD27-TAP) corrects nuclear localization of Rpb8-ECFP in Δbud27 mutant cells at 30°C when compared to the same strain containing an empty plasmid (pCM).RNA pols localisation. A) Deletion of Bud27 NES domain does not impair RNA pols nuclear localisation (Rpb8-ECFP). Δ(TIF)Click here for additional data file.Figure S3siRNA silencing of URI. URI mRNA accumulation was determined in human pulmonary fibroblast grown in GM medium at 37°C transfected with 40, 100 and 200 nM of siRNA(URI) heteroduplex. CONTROL cells were treated in the same conditions without siRNA heteroduplex.(TIF)Click here for additional data file.Figure S4RPB5 overexpression rescues nuclear RNA pols localisation. Immunocytochemistry experiments using antibodies against Rpa190-HA (anti-HA), Rpb1 (8WG16), and Rpc160-Myc (anti-Myc) in Δbud27 mutant cells with tagged Rpa190-HA (RNA pol I) and Rpc160-Myc (RNA pol III), at 30°C, transformed with a plasmid overexpressing RPB5 (pFL-RPB5), or with an empty vector (pFL).(TIF)Click here for additional data file.Table S1bud27 mutant containing functional tagged version of Rpb3 (Rpb3-TAP). The protein mixture obtained in each case was subjected to multidimensional protein identification technology (MudPIT) Rpb3-TAP purification from wild type and Δ(DOC)Click here for additional data file.


The serotonin transporter (SERT) belongs to the SLC6 family of neurotransmitter transporters, which mediate reuptake of previously released neurotransmitters from the synapse. Mutation of C-terminus residues RI607–608 to alanine results in intracellular retention of SERT . We subsUsing siRNAs to knock down SEC24 isoforms A–D in HeLa cells, we screened a series of double and truncation mutants generated along the C-terminus of SERT. HeLa cells were transfected with Sec24 siRNAs and, after 48 h, with YFP-tagged transporter plasmids. Functional effects of SEC24A–D knockdowns were determined by substrate uptake assays.Export of the IKAA-SERT mutant was not sensitive to knockdown of Sec24C. Remarkably, the closely related transporters for dopamine (DAT) and noradrenaline (NET), rely on Sec24D, and not C, for their ER export [The data imply that residue K610 and the equivalent residues in other transporters specify which SEC24 paralogue is recruited for ER export. These export signals work independently because a concatemer of SERT and GAT-1 is affected by depletion of both SEC24C and SEC24D.


A central challenge in designing RNA-Seq-based experiments is estimating We have conducted a systematic examination of how changes in the number of RNA-Seq reads per sample influences both profiling of a single bacterial transcriptome and the comparison of gene expression among samples. Our findings suggest that the number of reads typically produced in a single lane of the Illumina HiSeq sequencer far exceeds the number needed to saturate the annotated transcriptomes of diverse bacteria growing in monoculture. Moreover, as sequencing depth increases, so too does the detection of cDNAs that likely correspond to spurious transcripts or genomic DNA contamination. Finally, even when dozens of barcoded individual cDNA libraries are sequenced in a single lane, the vast majority of transcripts in each sample can be detected and numerous genes differentially expressed between samples can be identified.Our analysis provides a guide for the many researchers seeking to determine the appropriate sequencing depth for RNA-Seq-based studies of diverse bacterial species. In recent years, high throughput sequencing of cDNA libraries (RNA-Seq) has emerged as a powerful technology for profiling gene expression, discovering previously unannotated genes, and mapping transcriptome architecture in a wide variety of bacterial species -11. RNA-The relative abundances of individual transcripts in a bacterial transcriptome can differ by several orders of magnitude. In order to generate comprehensive transcriptome profiles using RNA-Seq one must therefore obtain a sufficiently large number of reads to detect those biologically relevant transcripts that comprise a relatively small proportion of the cDNA library. Detection and quantification of low abundance transcripts by RNA-Seq can be enhanced in two main ways. First, the total number of reads per library can be increased. Second, the proportion of reads representing rare transcripts can be increased by depleting abundant transcripts from total RNA and/or depleting cDNAs representing these abundant transcripts from cDNA libraries. This is often achieved by targeted removal of ribosomal RNA (rRNA), which comprises 80-95% of bacterial transcriptomes, from total RNA prior to cDNA library construction ,15.For many RNA-Seq-based projects, the budget for sequencing costs, and thus the total number of reads that can be obtained, is constrained. Thus, researchers designing RNA-Seq experiments must often determine the correct balance between sequencing depth (the number of reads per sample) and breadth (the number of samples sequenced). For some applications of RNA-Seq such as transcriptome mapping and annotation, the ability to detect rare transcripts is critical, and approaches such as the ones described above for increasing the total number of biologically relevant reads obtained per sample play a central role. For other applications of RNA-Seq breadth can often be more important than depth. Specifically, for experiments focused on comparing gene expression among various strains and/or growth conditions, the inclusion of more strains, timepoints, biological replicates, and/or growth conditions may be worth the tradeoff of lower depth per sample, as it may provide additional biological insights and/or statistical confidence that is more valuable than the ability to detect low abundance transcripts in each sample.E. coli and examined how reducing sequencing depth affects the ability to detect and quantify transcripts both within and between samples in diverse bacterial species.In recent years, methods for incorporating barcoded adapters into cDNA libraries have been developed that allow reads derived from up to several dozen samples to be sequenced in the same lane . This apEscherichia coli K-12 which was then depleted of rRNA using the RiboZero kit (Epicentere), converted to a strand-specific Illumina cDNA library as described [E. coli K-12 genome. Over 97% of these reads corresponded to properly mapped paired end reads, i.e. those corresponding to reads derived from opposite ends of the same cDNA mapping no more than 450 base pairs apart on the genome (the approximate maximum size of cDNAs in the library – see Methods). Properly mapped paired end reads were resolved into a single fragment by filling in the gap between them (if any). For pairs of reads that was not properly mapped, one read was discarded and the remaining reads along with unpaired reads were each treated as independent fragments. In total this dataset contained approximately 156 million aligned fragments with an average length of 159 nucleotides. rRNA depletion in this sample was nearly complete, with less than 0.15% of fragments aligning to rRNA-encoding genes  requires ~700 million reads ; howeverescribed , and seqThe proportion of annotated ORFs represented in this dataset was very high, with all but 2 of 4149 ORFs annotated in RefSeq covered by at least 1 fragment  between the fragment coverage of sense and antisense strands, even among highly expressed genes. In contrast, the correlation in the coverage of ORF sense and antisense strands was much higher (R2=0.83) when a similar rRNA-depleted E. coli cDNA library was not subjected to dUTP excision prior to amplification and sequencing. Thus, incomplete strand specificity in our libraries does not seem to have contributed significantly to the observed high coverage of antisense positions.As shown in Figure E. coli genome is transcribed than is suggested by current gene annotations. Indeed, several recent studies have demonstrated widespread transcription from the antisense strand of protein-encoding genes in diverse bacteria [Bacillus subtilis and E. coli K-12 suggest that many antisense RNAs derive from spurious transcription initiation or incomplete transcription termination and may not be functionally relevant [E. coli K-12 [Another explanation for the high coverage of antisense and intergenic positions observed is that a much higher proportion bacteria -23. Whilrelevant ,25. Thusoli K-12 ,26.E. coli genomic DNA (gDNA). While total RNA was subjected to 2 rounds of DNase treatment and no gDNA was detected following 40 rounds of PCR prior to cDNA synthesis, it is possible that removal of gDNA from our total RNA was not complete. Similarly, reagents used after DNase treatment in library construction may also have introduced low amounts of E. coli gDNA contamination.Finally, the nearly complete RNA-Seq read coverage of the genome could also reflect contamination of our cDNA libraries with a low amount of E. coli K-12 transcriptome but also yields numerous fragments aligning to very rare and potentially non-functional transcripts and/or to low-level contaminants introduced during library construction.Taken together our findings suggest that a sequencing depth of 156 million fragments is sufficient to saturate the E. coli genes was nearly complete in the 156M read dataset, the possibility of gDNA contamination raised concern that some of these genes were not actually transcribed. To better estimate the proportion of E. coli genes transcribed under the conditions tested, we devised an algorithm to subtract potential gDNA background from our RNA-Seq dataset based on the assumption that, unlike reads corresponding to cDNAs, the alignment of reads corresponding to gDNA would be uniformly distributed across the E. coli genome. As shown in Figure E. coli genes are expressed at least at low levels during exponential growth in rich media.While read coverage of annotated ibs toxic membrane proteins that may indeed not be expressed under normal growth conditions [rut operon involved in pyrimidine degradation [cit operon encoding components of an inactive citrate lyase [phn operon required for use of phosphonate and phosphite as phosphorous sources [E. coli K-12 in LB medium.Of the 100 ORFs to which no reads aligned following 1% subtraction, several are near the minimum size cutoff of cDNAs efficiently maintained during library construction. These include 4 of the 5 nditions   with those of a dataset of 15 million fragments sampled from the 156 million fragment dataset.To systematically assess how decreasing fragment counts per sample affects the comprehensiveness of gene expression profiles, we developed scripts that randomly sampled our 156 million fragment rRNA-depleted As shown in Figure E. coli transcripts expressed during log phase growth in LB. Moreover, they suggest that vast majority of the E. coli transcriptome can be detected under this growth condition even with datasets of only 5-10 million non-rRNA fragments.As shown in Figure E. coli K-12 cultures growing in LB, we repeated the analysis above with RNA-Seq data derived from log phase LB cultures of Mycobacterium tuberculosis  at the late exponential and early stationary phases. cDNA libraries corresponding to 2 biological replicates for each time point were subjected to multiplexed sequencing using Illumina HiSeq to yield 25-30 million fragments per sample. Data between biological replicates for each time point was were extremely well correlated (R2 of fragments/ORF = 0.99). To examine the impact of having fewer fragments on the results of differential expression analysis, we scaled down the counts of fragments per gene from each dataset while retaining the original values of relative gene expression.In addition to its utility in profiling the transcriptome of a single strain of interest, RNA-Seq is also a powerful tool for comparing gene expression among different strains and/or growth conditions. A recent study by Tarazona Seq data  but to dWe first used these sampled datasets to determine how changes in sequencing depth influenced the detection of transcripts in two independent biological replicates. As shown in Figure -3). As shown in Figure -3. Our findings indicate that when data from well-correlated biological replicates are included, 2-3 million fragments per sample enable a significant number of genes differentially expressed by 2-fold or more to be identified with high statistical significance.We next analyzed the full and sampled datasets with DESeq, a variance-analysis package that uses a model based on the negative binomial distribution to infer statistically significant differences in gene-expression from RNA-Seq data . Based oV. cholerae growing in M9 minimal medium or isolated from the cecal fluid of 2 orally infected infant rabbits [2=0.69). Finally, the total number of non-rRNA fragments for these datasets was between 4 and 6 million, significantly less than in the EDL933 datasets.The ability to reliably identify differentially expressed genes by RNA-Seq is affected by a variety of factors aside from total sequencing depth that can vary significantly from one experiment to another, including the number of biological replicates included and the variation between them, the average abundance of differentially expressed genes, and the magnitude of their differential expression under the conditions tested. We therefore repeated the analysis above with RNA-Seq data that were distinct in several ways from the EDL933 data. Specifically, these data were derived from V. cholerae and EDL933 datasets on the ability to detect differentially expressed genes was very similar  even with a reduction of fragments to 2-3 million per sample. These included all 16 of the major V. cholerae colonization or virulence factors identified as induced in the rabbit when the full datasets were compared [-3) even when the total number of non-rRNA fragments was reduced 100-fold to 40,000-60,000 total fragments. While it is not possible to accurately simulate how changes in depth will affect RNA-Seq comparative gene expression analyses in all cases, our findings indicate that in diverse species and growth conditions and even with relatively low correlation between biological replicates, 2-3 million fragments per sample enable a significant number of genes differentially expressed by 2-fold or more to be identified with high statistical significance.Despite these numerous differences, the impact of reducing the number of fragments in the r Figure . ImportaE. coli exceeds 50 million, detection of biologically relevant transcripts all but ceases and much of the additional coverage gained appears to represent very rare transcriptional events and/or gDNA contamination. We also found that when RNA-Seq data from biological replicates is available, differential expression of numerous genes can be detected with high statistical significance even when the number of fragments per sample is reduced to 2-3 million.We have conducted a systematic analysis of how changes in sequencing depth affect analysis of bacterial RNA-Seq data, both for profiling gene expression in a single sample and for comparing gene expression among different strains and/or growth conditions. Our findings suggest that 5-10 million non-rRNA fragments are sufficient to detect all but a few of the most low expressed genes in diverse bacteria growing under a variety of conditions. Moreover, we found that when the number of non-rRNA fragments in bona fide transcripts. Alternatively, the number and diversity of growth conditions included in the analysis can be increased with the expectation that, while the number of reads per sample will be decreased, numerous transcripts whose abundance is low under one condition will be more highly expressed and thus easier to detect under another condition. For applications aimed at discovery of a relatively small number of previously unannotated genes to be subjected to experimental validation and/or functional characterization, lower sequencing depth can provide sufficient sensitivity. Indeed, a depth of 4 million non-rRNA fragments was sufficient for identification of several dozen previously unannotated ncRNAs in V. cholerae[-5). Thus, our findings suggest that for many RNA-Seq based studies in bacteria, the number of fragments needed to profile gene expression in a single rRNA-depleted sample isolated from a bacterial monoculture is far less than that produced in a single Illumina HiSeq lane. Indeed, our findings suggest that at a certain point increased sequencing depth may actually be detrimental to the accurate mapping of biologically relevant transcripts, yielding reads that likely represent contaminants in the cDNA library or the products of spurious transcriptional events.The optimal sequencing depth for an RNA-Seq based study will vary considerably based on the scientific objective of that study. For applications requiring a comprehensive transcriptome profile, coverage exceeding 10 million fragments per sample may be needed, with the understanding that increasing depth can lead to detection of sequences that may not represent  cholerae. SimilarA HiSeq lane typically produces about 150 million paired end reads under current run conditions. Thus, multiplexing 15-30 samples per lane will yield the 5-10 million reads per sample that are sufficient for most applications of bacterial RNA-Seq. Indeed, our findings suggest that for studies of differential gene expression, even significantly higher levels of multiplexing result in relatively modest decreases in sensitivity. For these types of studies, the added biological information provided by the inclusion of more strains, growth conditions, and/or biological replicates may outweigh this loss of sensitivity for detecting transcriptional changes in each pairwise comparison of samples. Our findings also suggest that for studies in which only a few samples are to be sequenced in a single lane, a sufficient number of reads may be obtained for samples that are not depleted of rRNA and thus the time and cost associated with rRNA-depletion may not be justified. Finally, for studies involving only one or two samples, such as pilot or proof-of-principle experiments, lower throughput platforms such as Illumina MiSeq platform may be more appropriate than the HiSeq platform. MiSeq yields only about 7.5 million paired end reads per lane with a only a slightly lower reagent cost than a lane of HiSeq but produces data in a fraction of the time needed for a HiSeq run, making it a good option for those seeking to quickly obtain profiles of gene expression in only a few rRNA-depleted samples.The analysis we conducted was largely limited to data derived from single bacterial strains grown in culture. However, RNA-Seq is increasingly being used to study the transcriptomes of bacteria growing in animal hosts and/or as part of complex bacterial communities. Samples isolated from animal models are often contaminated with a large amount of host RNA. In RNA derived from microbial communities, transcripts corresponding to particular strains of interest will often be greatly outnumbered by those expressed by the numerous other members of the community. Thus, in RNA-Seq data representing mixed samples, the number of reads corresponding to transcripts of interest can be orders of magnitude lower than in data derived from a homogeneous bacterial culture. Using RNA-Seq to unravel the dynamics of bacterial gene expression in these complex and biologically relevant samples will therefore require significantly greater sequencing depth per sample, a robust depletion of bacterial rRNA, host rRNA, and host mRNA, and/or enrichment for transcripts of interest through methods such as hybrid capture.We have conducted a systematic analysis of how changes in sequencing depth influence the profiling and comparison of transcriptomes by RNA-Seq in diverse bacterial species and growth conditions. Our findings provide a guide for determining the appropriate sequencing depth for a wide variety of RNA-Seq-based studies of bacterial gene expression.M. tuberculosis RNA included bead beating during incubation with TRIzol [E. coli and EDL933) and Gram-negative Kit (for M. tuberculosis) (Epicentre) according to the manufacturer’s protocol. mRNA-enriched RNA isolated using Zymo RNA Clean & Concentrator columns (Zymo Research) and treated with DNase using the TURBO DNA-free kit (Ambion) according to the manufacturer's protocol. The RNA was then fragmented in a reaction with 5X Fragmentation Buffer (Affymetrix) heated at 80°C for 6 minutes and purified using the Zymo RNA Clean & Concentrator columns (Zymo Research).RNA was isolated by incubation by TRIzol (Invitrogen) followed by passage through Direct-zol columns (Zymo Research). Isolation of h TRIzol . Total RE. coli DNA polymerase I (NEB). This reaction was incubated at 16°C for 2 hrs then placed on ice and terminated with 10 ul of 0.5 M EDTA. cDNA was then isolated from this reaction using the MinElute PCR Purification Kit (Qiagen).Unless otherwise indicated, all reagents in this section were obtained from Invitrogen. For first strand cDNA synthesis, RNA was incubated with random hexamers at 70°C for 10 minutes and then chilled on ice. The primer and RNA template mix was then added to 5X FS Buffer, 0.1 M DTT, 10 mM dNTP mix, Actinomycin D (Sigma-Aldrich), Superase-in (Ambion), and SuperScript III. This reaction was incubated at 25°C for 10 minutes and at 55°C for 1 hour, then chilled for 5 minutes on ice and cleaned up using Zymo RNA Clean & Concentrator (Zymo Research). The second strand cDNA synthesis reaction contained the product of the first strand synthesis reaction, 5X FS Buffer, 5X SS Buffer, 0.1 M DTT, 10 mM dUTP mix (Affymetrix/USB), RNase H, DNA Ligase (NEB), and cDNA fragments were end-repaired and phosphorylated, followed by adenylation of 3′ends and adapter ligation as described  with theReads were aligned to RefSeq reference genomes . Differentially expressed genes were identified using the feature-assigned fragment counts for each replicate as input to the DESeq software .Genome sequence coverage by RNA-Seq alignments was computed using a custom PERL script, where the strand-specific nucleotide coverage (C) was incremented at each nucleotide position spanned by a read or across the range covered by the boundaries of an RNA-Seq fragment inferred from a pair of properly mated paired end reads. Background subtraction assuming a given percent of genomic DNA contamination (pctBkg) was performed as follows. The total strand-specific coverage was computed by summing strand-specific nucleotide-level coverage (Csum) observed across the genome. The expected nucleotide-level coverage due to genomic DNA contamination (Cbkg) was computed as:The effective nucleotide-level background-subtracted coverage (Ceff) values were computed as follows:cDNA: Complementary DNA synthesized from RNA; RNA-Seq: High throughput sequencing of cDNA libraries; ORF: Open reading frame; ncRNA: Non-coding RNA; rRNA: Ribosomal RNA; tRNA: Transfer RNA; gDNA: Genomic DNA.The author’s declare that they have no competing interests.E. coli and M. tuberculosis cDNA Illumina libraries. BJH and JL analyzed data. JL, BWB, and CN directed the project and coordinated the research. JL wrote the paper with input from BJH, BWB, and CN. All authors read and approved the final manuscript.MC constructed Table S1. Refseq accession numbers for strains included in this study.Click here for fileTable S2. Reads per annotated ORF in 156M fragment data set before and after background subtraction.Click here for fileFigure S1. Correlation of coverage of the sense and antisense strands of annotated ORFs.Click here for file


Staphylococcus epidermidis is the major bacterial species found in biofilm-related infections on indwelling medical devices. Obtaining high quality mRNA from biofilms is crucial to validate the transcriptional measurements associated with the switching to the biofilm mode of growth. Therefore, we selected three commercially available RNA extraction kits with distinct characteristics, including those using silica membrane or organic extraction methods, and enzymatic or mechanical cell lysis, and evaluated the RNA quality obtained from two distinct S. epidermidis bacterial biofilms.Microbial biofilms are communities of bacteria adhered to a surface and surrounded by an extracellular polymeric matrix. Biofilms have been associated with increased antibiotic resistance and tolerance to the immune system. S. epidermidis strain. The results demonstrated that the kit based on mechanical lysis and organic extraction (FastRNA® Pro Blue) was the only one that was able to isolate pure and large quantities of RNA. Normalized expression of the icaA virulence gene showed that RNA extracted with PureLink™ had a significant lower concentration of icaA mRNA transcripts than the other kits tested.RNA extracted using the different kits was evaluated for quantity, purity, integrity, and functionally. All kits were able to extract intact and functional total RNA from the biofilms generated from each ® Pro Blue was the best option for both S. epidermidis biofilms used.When working with complex samples, such as biofilms, that contain a high content extracellular polysaccharide and proteins, special care should be taken when selecting the appropriate RNA extraction system, in order to obtain accurate, reproducible, and biologically significant results. Among the RNA extraction kits tested, FastRNA Staphylococcus epidermidis, a normal inhabitant of normal human skin and mucosa, has recently emerged as a leading cause of biofilm-related infections, particularly, in patients with indwelling medical devices [S. epidermidis biofilm matrix, seems to interfere with RNA extraction methods making bacterial cell lysis and nucleic acid purification difficult, and the purified RNA may still contain inhibitory substances [S. epidermidis biofilms. The selected kits were based on distinct procedures and properties: FastRNA® Pro Blue  uses mechanical and chemical lysis together with organic extraction; PureZOL™ RNA isolation reagent  uses chemical lysis with organic extraction, while PureLink™ RNA Mini Kit  uses enzymatic lysis and silica membrane extraction. devices ,2 due to devices . The qua devices ,5. Howev devices ,7. Curre devices -10. Biof devices . Polysacbstances ,13. TherS. epidermidis biofilms from two S. epidermidis strains with different genetic backgrounds  were incounting . Subsequg for 6 min at 4°C. This procedure did not reduced cell viability as determined by CFU counting [Biofilm total biomass, protein, and polysaccharide matrix content was determined as described previously . Brieflycounting . The supcounting  . The absorbance ratios A260/A280 were used as indicators of protein contamination and A260/A230 as indicators of polysaccharide, phenol, and/or chaotropic salts contamination [Total RNA was isolated according the manufacturer's instructions, with the following modification, when appropriate: cell lysis was performed using 15 mg/mL of lysozyme for 60 min at 37°C with. This optimization increased the yield of total RNA 2-4-fold (data not shown). The final total RNA fraction was obtained by eluting or suspending the RNA in 45 μL of DEPC-treated water. To digest contaminating DNA, DNase  treatment was performed by adding 5 μL (10×) of reaction buffer with MgClmination . The inticaA, a well-known virulence gene of S. epidermidis were designed using the Primer3 software [icaA and 95 ± 7% for 16S rRNA). To ensure the absence of genomic DNA contamination, a negative control was included in the reverse transcriptase reaction. The cycle threshold detection of each gene was determined using the standards parameters of the software. The melting curves were evaluated to ensure the absence of unspecific products and primer dimer formation. The expression of icaA was determined by using the delta Ct method (2ΔCt), a variation of the Livak method, where ΔCt = Ct (reference gene)-Ct (target gene). The data analysis was based on two to four independent experiments.To determine if the extracted RNA was functional, 40 ng of total RNA was reverse transcribed using an iScript™ cDNA Synthesis Kit (Bio-Rad) following the manufacturer's instructions. Real time PCR (qPCR) was performed to quantify the mRNA transcripts. Primers (Invitrogen), specific for 16S rRNA, reference gene, and software   , the only kit with a polysaccharides level (A260/A230) above 1.8 was FastRNA® Pro Blue (P < 0.01 unpaired t-test). Interestingly, significant differences were found in the total RNA quantity of the two strains of S. epidermidis used: while using FastRNA® Pro Blue strain 1457 yielded more RNA than strain M187, the opposite occurred in all the remaining kits. These differences are probably related with the strain specific ability to form biofilm and also with the composition of the biofilm matrix  and polysaccharide (P < 0.05 unpaired t-test) content than strain M187. As strain 1457 had a thicker biofilm, it seems reasonable to assume that the total number of cells available per biofilm would be higher, even taking in consideration that the majority of biofilm is composed of matrix and not bacteria [9 CFU/mL, strain M187 had 6.3×108 CFU/mL. Thus, the initial amount of bacterial cells used for the RNA extraction seams to influence the total RNA quantity, as also described in the manufacturer guidelines. However, the relationship between the amount of sample and the RNA yield is not linear and each kit normally has an optimal range of sample concentration . Nour et al. [® Pro Blue while in the remaining kits this was not the case. Interestingly, with strain M187, where the matrix presented lower levels of protein and polysaccharides, PureZOL™ and PureLink™ were more efficient as compared with strain 1457. Since FastRNA® Pro Blue was the only kit using mechanical lysis and had the highest RNA extraction performance, we tried to perform RNA extraction with PureZOL™ and PureLink™ preceded with the mechanical lysis step present on FastRNA® Pro Blue. Therefore, each biofilm was resuspended in the lysis buffer of the respective kit, but the lysis was completed mechanically following FastRNA® Pro Blue instructions. Interestingly, this modification did not significantly increased the RNA yield (P > 0.10 unpaired t-test) (data not show). This suggests that the high RNA yield obtained with FastRNA® Pro Blue depends not only on the mechanical lysis, but also on the chemistry. The buffer used for FastRNA® Pro Blue lysis procedure contains phenol which is known to induce cell lysis [The success of any RNA-based analysis depends on the quantity, purity, and integrity of the RNA ,5. RNA qt) Table . Comparibacteria . We havenation A20/A280, tnation A20/A280, tll lysis , thus yiBesides the yield and purity, the extracted RNA should be intact and functional, in order to proper analyze the quantification of gene expression . RNA inticaA were significantly lower (P < 0.01 unpaired t-test) when using the RNA obtained from the PureLink™. This significant difference elucidates the importance of selecting the proper RNA extraction kit for the biological system being studied. It has been point-out that qPCR requires low amounts of total RNA, as when compared with microarrays [A previous study regarding the optimization of complementary DNA (cDNA) synthesis using commercially available kits, showed that in order to obtain reliable mRNA quantification at least 40 ng of total RNA is necessary . Thus, froarrays . However260/280 and A260/230 ratios are just indicators of possible contaminants in the RNA sample. While some of the contaminants can interfere in the downstream applications, it seems that more than the concentration of contaminants, the nature of contaminants will impair RNA quantification. FastRNA® Pro Blue showed the best results, while PureLink™ RNA mini kit was the worst kit for S. epidermidis biofilm samples.Observing the overall results it can be seen that depending on the RNA extraction kit used, the quantification of mRNA transcripts can be impaired. Interestingly, of all the parameters tested, RNA purity seemed to have a lower impact in inhibiting RNA quantification. The AWhen working with complex samples, such as biofilms, that contain a high content extracellular polysaccharide and proteins, special care should be taken when selecting the appropriate RNA extraction system, in order to obtain accurate, reproducible, and biologically significant results. Testing different systems and probing for well described gene expression conditions might elucidate some less apparent pitfalls of RNA extraction kits.BCA: Bicinchoninic Acid; cDNA: complementary DNA; CFU: Colony-forming unit: Ct: cycle threshold; mRNA: messenger RNA; OD: optical density: qPCR: Real time PCR; TSB: Tryptic Soy Broth.AF carried-out all of the RNA related experiments and drafted the manuscript. LDRM performed the biofilm formation and characterization analysis and aided in drafting the manuscript. NC conceived the study, participated in its design and coordination, and aided in drafting the manuscript. All authors read and approved the final manuscript.The authors declare that they have no competing interests.