Primer For Reverse Transcription Research Articles

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BioTechniquesVol. 61, No. 2 BioSpotlight / CitationsOpen AccessBioSpotlight / CitationsPatrick C.H. Lo & Nathan S. BlowPatrick C.H. LoSearch for more papers by this author & Nathan S. BlowSearch for more papers by this authorPublished Online:16 Mar 2018https://doi.org/10.2144/000114439AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInReddit A reality check for dsRNAsA number of popular methods and commercial kits for single-step isolation of high-quality RNA use a concentrated solution of guanidinium thiocyanate (GuSCN)–a powerful chaotropic agent—to denature cellular proteins and inactivate RNases. Besides acting as a robust protein denaturant, a less well-known effect of concentrated GuSCN solutions is to promote DNA—DNA, RNA—DNA, and RNA—RNA hybridization. In this issue of BioTechniques, Triin Mölder and Mart Speek at the Tallinn University of Technology provide evidence suggesting that use of concentrated GuSCN during isolation of cellular RNA causes the formation of double-stranded RNAs (dsRNAs) from complementary single-stranded RNAs (ssRNAs). The authors demonstrated that incubation in a concentrated GuSCN solution of low concentrations of two in vitro—transcribed RNAs with a region of complementarity catalyzed their hybridization into dsRNA, in the absence or presence of total cellular RNA. This suggests the need to carefully verify that any putative novel dsRNAs identified from RNA samples isolated by GuSCN extraction are not actually artifacts caused by this effect.See “Accelerated RNA—RNA hybridization by concentrated guanidinium thiocyanate solution in single-step RNA isolation”Indenting multiple cellsStudies of the response of cells to mechanical force transduction have relied on instruments such as atomic force microscopes (AFMs) or cytoindentors, but these tools can only indent single cells one-by-one, which makes the collection of data from a sufficient number of cells a tedious and time-consuming affair. To overcome this problem, researchers at McGill University led by Claire Brown and Sabrina Leslie describe in this month's issue their development of a high-throughput, parallelized cytoindentor for the simultaneous local compression of multiple live cells. They modified their previously described convex lens—induced confinement (CLiC) set-up by adhering a film patterned with microposts to a microscope lens. This lens is then gradually lowered onto multiple cells maintained in a stage-top incubator to indent micrometer-sized areas on each cell while allowing imaging of those cells. By functionalizing the micropost arrays with an extracellular matrix protein such as fibronectin, it was also possible to pull on the cells. This novel parallelized cytoindentor will greatly facilitate studies of mechanobiology in living cells.See “Parallelized cytoindentation using convex micropatterned surfaces”Protein interactions: limiting diffusion on arraysProtein arrays are important tools for those exploring protein—protein interactions on a large scale. These arrays can be spotted with purified proteins, or cell-free protein synthesis can be performed on the arrays. Protein synthesis approaches such as the nucleic acid programmable protein array (NAPPA) streamline the building of large arrays, but they also produce large, diffuse protein spots that limit array density. Yazaki et al. solve this problem with their newly devised HaloTag- NAPPA technology, which produces higher-density protein spotting. To accomplish this, they exchanged the C-terminal GST fusion used in the original NAPPA for a HaloTag fusion that can be irreversibly bound to ligands co-spotted on the array. HaloTag-NAPPA captured synthesized proteins with significantly less diffusion compared to GST fusion proteins. To test their method, the authors spotted 12,000 Arabidopsis clones onto arrays, synthesized proteins, and then examined protein— protein interactions with 38 plant transcription factors. The new approach identified novel interactions with high sensitivity and low background.Yazaki, J. et al. 2016. Mapping transcription factor interactome networks using HaloTag protein arrays. Proc. Natl. Acad. Sci. USA. DOI: 10.1073/pnas.1603229113Gene expression profiling: it's all about locationProfiling gene expression is a powerful approach for understanding basic biology and human disease. Now, Stahl et al. introduce a new method that could significantly expand the scope of gene expression profiling by adding spatial resolution within tissue samples. In “spatial transcriptomics,” histological samples are placed onto slides with reverse transcription primers possessing positional barcodes immobilized on the slide surface. Following permeabilization, reverse transcription reagents are placed onto the tissues, where cDNA is synthesized and left coupled to the oligonucleotides on the slide. Once the tissue is enzymatically removed, the cDNAs are collected and sequenced, akin to traditional gene expression profiling. After sequencing, the locations of cDNA transcripts can be mapped back to the tissue using the incorporated positional barcodes. By adding only a few additional steps, scientists will now be able to obtain spatial information by transcriptional profiling, thus greatly enhancing gene expression data sets.Stahl, P.L., et al. 2016. Visualization and analysis of gene expression in tissue sections by spatial transcriptomics. Science. 353(6294):78-82.FiguresReferencesRelatedDetails Vol. 61, No. 2 Follow us on social media for the latest updates Metrics History Published online 16 March 2018 Published in print August 2016 Information© 2016 Author(s)PDF download

Targeted reduction of highly abundant transcripts using pseudo-random primers.

Transcriptome studies based on quantitative sequencing can estimate levels of gene expression by measuring target RNA abundance in sequencing libraries. Sequencing costs are proportional to the total number of sequenced reads, and in order to cover rare RNAs, considerable quantities of abundant and identical reads are needed. This major limitation can be addressed by depleting a proportion of the most abundant sequences from the library. However, such depletion strategies involve either extra handling of the input RNA sample or use of a large number of reverse transcription primers, termed not-so-random (NSR) primers, which are costly to synthesize. Taking advantage of the high tolerance of reverse transcriptase to mis-prime, we found that it is possible to use as few as 40 pseudo-random (PS) reverse transcription primers to decrease the rate of undesirable abundant sequences within a library without affecting the overall transcriptome diversity. PS primers are simple to design and can be used to deplete several undesirable RNAs simultaneously, thus creating a flexible tool for enriching transcriptome libraries for rare transcript sequences.

Compiling Multicopy Single-Stranded DNA Sequences from Bacterial Genome Sequences.

A retron is a bacterial retroelement that encodes an RNA gene and a reverse transcriptase (RT). The former, once transcribed, works as a template primer for reverse transcription by the latter. The resulting DNA is covalently linked to the upstream part of the RNA; this chimera is called multicopy single-stranded DNA (msDNA), which is extrachromosomal DNA found in many bacterial species. Based on the conserved features in the eight known msDNA sequences, we developed a detection method and applied it to scan National Center for Biotechnology Information (NCBI) RefSeq bacterial genome sequences. Among 16,844 bacterial sequences possessing a retron-type RT domain, we identified 48 unique types of msDNA. Currently, the biological role of msDNA is not well understood. Our work will be a useful tool in studying the distribution, evolution, and physiological role of msDNA.

Mapping RNA Structure In Vitro with SHAPE Chemistry and Next-Generation Sequencing (SHAPE-Seq).

Mapping RNA structure with selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) chemistry has proven to be a versatile method for characterizing RNA structure in a variety of contexts. SHAPE reagents covalently modify RNAs in a structure-dependent manner to create adducts at the 2'-OH group of the ribose backbone at nucleotides that are structurally flexible. The positions of these adducts are detected using reverse transcriptase (RT) primer extension, which stops one nucleotide before the modification, to create a pool of cDNAs whose lengths reflect the location of SHAPE modification. Quantification of the cDNA pools is used to estimate the "reactivity" of each nucleotide in an RNA molecule to the SHAPE reagent. High reactivities indicate nucleotides that are structurally flexible, while low reactivities indicate nucleotides that are inflexible. These SHAPE reactivities can then be used to infer RNA structures by restraining RNA structure prediction algorithms. Here, we provide a state-of-the-art protocol describing how to perform in vitro RNA structure probing with SHAPE chemistry using next-generation sequencing to quantify cDNA pools and estimate reactivities (SHAPE-Seq). The use of next-generation sequencing allows for higher throughput, more consistent data analysis, and multiplexing capabilities. The technique described herein, SHAPE-Seq v2.0, uses a universal reverse transcription priming site that is ligated to the RNA after SHAPE modification. The introduced priming site allows for the structural analysis of an RNA independent of its sequence.

A Comprehensive Analysis of Primer IDs to Study Heterogeneous HIV-1 Populations

Determining the composition of viral populations is becoming increasingly important in the field of medical virology. While recently developed computational tools for viral haplotype analysis allow for correcting sequencing errors, they do not always allow for the removal of errors occurring in the upstream experimental protocol, such as PCR errors. Primer IDs (pIDs) are one method to address this problem by harnessing redundant template resampling for error correction. By using a reference mixture of five HIV-1 strains, we show how pIDs can be useful for estimating key experimental parameters, such as the substitution rate of the PCR process and the reverse transcription (RT) error rate. In addition, we introduce a hidden Markov model for determining the recombination rate of the RT PCR process. We found no strong sequence-specific bias in pID abundances (the same RT efficiencies as compared to commonly used short, specific RT primers) and no effects of pIDs on the estimated distribution of the references viruses.

Crystal Structure of the Human tRNA m1A58 Methyltransferase–tRNA3Lys Complex: Refolding of Substrate tRNA Allows Access to the Methylation Target

Human tRNA3Lys is the primer for reverse transcription of HIV; the 3′ end is complementary to the primer-binding site on HIV RNA. The complementarity ends at the 18th base, A58, which in tRNA3Lys is modified to remove Watson–Crick pairing. Motivated to test the role of the modification in terminating the primer-binding sequence and thus limiting run-on transcription, we asked how the modification of RNA could be accomplished. tRNA m1A58 methyltransferase (m1A58 MTase) methylates N1 of A58, which is buried in the TΨC-loop of tRNA, from cofactor S-adenosyl-l-methionine. This conserved tRNA modification is essential for stability of initiator tRNA in Saccharomyces cerevisiae. Reported here, three structures of human tRNA m1A58 MTase in complex with human tRNA3Lys and the product S-adenosyl-l-homocysteine show a dimer of heterodimers in which each heterodimer comprises a catalytic chain, Trm61, and a homologous but noncatalytic chain, Trm6, repurposed as a tRNA-binding subunit that acts in trans; tRNAs bind across the dimer interface such that Trm6 from the opposing heterodimer brings A58 into the active site of Trm61. T-loop and D-loop are splayed apart showing how A58, normally buried in tRNA, becomes accessible for modification. This result has broad impact on our understanding of the mechanisms of modifying internal sites in folded tRNA. The structures serve as templates for design of inhibitors that could be used to test tRNA m1A58 MTase's impact on retroviral priming and transcription.

On the primer binding site mutation that appears and disappears during HIV and SIV replication.

A recent study by Fennessey et al. (Retrovirology 12:49, 2015) described the optimization of a popular SIV clone by removal of four suboptimal point mutations. One of these mutations is present in a non-coding part of the viral genome and is probed in that study in more detail because of some fascinating properties. This primer binding site (PBS) mutation reverts rapidly to the wild-type sequence, which the authors interpret as indicating that this mutation exerts a profound fitness impact. The authors proposed the involvement of a cellular DNA repair mechanism in the reversion. Furthermore, it was suggested that premature termination of reverse transcription can explain why some of the viral progeny still contained the mutant sequence. However, we argue that all these special properties are a direct consequence of the unique nature of the viral PBS motif. The PBS binds the tRNA primer for reverse transcription and the viral progeny inherits either the sequence of the cellular tRNA or the PBS sequence of the viral RNA genome. The presence of a variant tRNA species explains the rapid appearance and disappearance of a variant PBS sequence.

Direct identification and discernment of Mycobacterium avium and Mycobacterium intracellulare using a real-time RNA isothermal amplification and detection method

The purpose of this work was to establish a real-time simultaneous amplification and testing method for identification and discernment of Mycobacterium avium and Mycobacterium intracellulare (SAT-MAC assay) and to evaluate the efficiency with which this method can detect isolated strains and clinical sputum specimens. The specific 16S rRNA sequences of M.avium and M.intracellulare were used as targets to design RNA probes and a reverse transcription primer containing T7 promoter. RNA isothermal amplification and real-time fluorescence detection were performed at 42°C. SAT-MAC assay, culture tests on Lowenstein-Jensen (L-J) culture medium and PCR-sequencing were used to test the clinical isolated strains and sputum specimens. The limit of detection (LOD) of M.avium and M.intracellulare by SAT-MAC was found to be 30CFU/mL and 20CFU/mL. SAT-MAC showed high specificity in 21 species of mycobacteria standard strains and 5 species of non-mycobacteria bacteria. Using PCR-sequencing as the reference method, both rates of SAT-MAC assay for identifying M.avium and M.intracellulare from clinical isolates were 100% (259/259). Consistent with the results of L-J culture combined PCR-sequencing, the coincidence rate of SAT-MAC assay in clinical sputum specimens was 100% (369/369) for M.avium and 99.19% (366/369) for Mycobacterium intracellular. The SAT-MAC assay can identify and distinguish M.avium and M.intracellulare rapidly and accurately. It may be suitable for use in clinical microbiology laboratories.

P40. MIR-21 and mir-221 overexpression in placental tissue of preeclamptic patients

Introduction Preeclampsia is a hypertensive disorder potentially leading to severe complications in mother and the fetus. Etiology of preeclampsia is multifactorial including partially genetic and immunological pathways; the process of apoptosis is also involved in the development of preeclampsia. MicroRNAs (miRNAs) - small noncoding molecules- are emerging as critical regulators of biological function (including apoptosis), and alterations to the miRNAome have been described in the context of pregnancy and PE. Objectives Goal of this work was to assess expression of pre-selected miRNAs in preeclamptic placental samples and comparison with samples from physiological pregnancies. We designed a 96-well reaction plate to analyze 21 miRNAs in duplicates described to be involved in the apoptosis regulation and expressed in placenta: pro- apoptotic miR-1, let-7c, let-7g, mir-200c, mir-143, mir-205, mir-122, mir-409-3p, mir-449, mir-708, mir-149, mir-204,mir-133 and anti-apoptotic : mir-214, mir221, mir 222, and miRNAs with both the anti-apoptotic and apoptotic targets mir29a and mir29c. Methods The normalization was performed against RNU44. The miRNAs were extracted from placental tissue obtained after delivery from both preeclamptic women and healthy controls. After stem-loop primer reverse transcription in one tube, the samples were analyzed on the plates and evaluated by delta Ct algorithm. The aberrantly expressed miRNAs were subsequently validated by TaqMan MicroRNA Assay by relative quantification with the standard curves. Results Overexpression of miRNA was observed in mir-122, mir-21, mir-221, mir-29a, mir-29c and mir-449. The validation was performed on 13 preeclamptic placental samples and 6 placental samples from physiological pregnancies; significant overexpression of mir-21 and mir-221 with p =0.0055 (CI 95% 25.2–123.47) and p =0.0047 (CI 95% 12.16–57.20) was observed, respectively. Conclusion Analysis of miRNA deregulation can help in the identification of deregulated signaling in preeclamptic placentas for detection of new, potentially usable biomarkers. Even though authors would like to point out the necessity of validation on a larger sample cohort.

Combined in vitro transcription and reverse transcription to amplify and label complex synthetic oligonucleotide probe libraries.

Oligonucleotide microarrays allow the production of complex custom oligonucleotide libraries for nucleic acid detection-based applications such as fluorescence in situ hybridization (FISH). We have developed a PCR-free method to make single-stranded DNA (ssDNA) fluorescent probes through an intermediate RNA library. A double-stranded oligonucleotide library is amplified by transcription to create an RNA library. Next, dye- or hapten-conjugate primers are used to reverse transcribe the RNA to produce a dye-labeled cDNA library. Finally the RNA is hydrolyzed under alkaline conditions to obtain the single-stranded fluorescent probes library. Starting from unique oligonucleotide library constructs, we present two methods to produce single-stranded probe libraries. The two methods differ in the type of reverse transcription (RT) primer, the incorporation of fluorescent dye, and the purification of fluorescent probes. The first method employs dye-labeled reverse transcription primers to produce multiple differentially single-labeled probe subsets from one microarray library. The fluorescent probes are purified from excess primers by oligonucleotide-bead capture. The second method uses an RNA:DNA chimeric primer and amino-modified nucleotides to produce amino-allyl probes. The excess primers and RNA are hydrolyzed under alkaline conditions, followed by probe purification and labeling with amino-reactive dyes. The fluorescent probes created by the combination of transcription and reverse transcription can be used for FISH and to detect any RNA and DNA targets via hybridization.

Strand-Specific Quantitative Reverse Transcription-Polymerase Chain Reaction Assay for Measurement of Arenavirus Genomic and Antigenomic RNAs.

Arenaviruses are bi-segmented, single-stranded RNA viruses that cause significant human disease. The manner in which they regulate the replication of their genome is not well-understood. This is partly due to the absence of a highly sensitive assay to measure individual species of arenavirus replicative RNAs. To overcome this obstacle, we designed a quantitative reverse transcription (RT)-PCR assay for selective quantitation of each of the lymphocytic choriomeningitis virus (LCMV) genomic or antigenomic RNAs. During the course of assay design, we identified a nonspecific priming phenomenon whereby, in the absence of an RT primer, cDNAs complementary to each of the LCMV replicative RNA species are generated during RT. We successfully circumvented this nonspecific priming event through the use of biotinylated primers in the RT reaction, which permitted affinity purification of primer-specific cDNAs using streptavidin-coated magnetic beads. As proof of principle, we used the assay to map the dynamics of LCMV replication at acute and persistent time points and to determine the quantities of genomic and antigenomic RNAs that are incorporated into LCMV particles. This assay can be adapted to measure total S or L segment-derived viral RNAs and therefore represents a highly sensitive diagnostic platform to screen for LCMV infection in rodent and human tissue samples and can also be used to quantify virus-cell attachment.

Genome-wide target site triplication of Alu elements in the human genome

Alu elements are the most successful short interspersed elements in primate genomes and their retrotransposition is a major source of genomic expansion. Alu elements integrate into genomic regions through target-site primed reverse transcription, which generates target site duplications (TSDs). Unexpectedly, we have identified target site triplications (TSTs) at some loci, where two Alu elements in tandem share one direct repeat. Thus, the three copies of the repeat are present. We located 212 TST loci in the human genome and examined 25 putative human-specific TST loci using PCR validation. As a result, 12 human-specific TST loci were identified. These findings suggest that unequal homologous recombination between TSDs can lead to TST. Through this mechanism, the copy number of Alu elements could have increased in primate genomes without new Alu retrotransposition events. This study provides new insight into the augmentation of Alu elements in the primate genome.

Surveillance and cleavage of eukaryotic tRNAs.

Beyond their central role in protein synthesis, transfer RNAs (tRNAs) have many other crucial functions. This includes various roles in the regulation of gene expression, stress responses, metabolic processes and priming reverse transcription. In the RNA world, tRNAs are, with ribosomal RNAs, among the most stable molecules. Nevertheless, they are not eternal. As key elements of cell function, tRNAs need to be continuously quality-controlled. Two tRNA surveillance pathways have been identified. They act on hypo-modified or mis-processed pre-tRNAs and on mature tRNAs lacking modifications. A short overview of these two pathways will be presented here. Furthermore, while the exoribonucleases acting in these pathways ultimately lead to complete tRNA degradation, numerous tRNA-derived fragments (tRFs) are present within a cell. These cleavage products of tRNAs now potentially emerge as a new class of small non-coding RNAs (sncRNAs) and are suspected to have important regulatory functions. The tRFs are evolutionarily widespread and created by cleavage at different positions by various endonucleases. Here, we review our present knowledge on the biogenesis and function of tRFs in various organisms.

Validation of a multiplex reverse transcription and pre-amplification method using TaqMan(®) MicroRNA assays.

Since the discovery of microRNAs (miRNAs), different approaches have been developed to label, amplify and quantify miRNAs. The TaqMan® technology, provided by Applied Biosystems (ABIs), uses a stem-loop reverse transcription primer system to reverse transcribe the RNA and amplify the cDNA. This method is widely used to identify global differences between the expression of 100s of miRNAs across comparative samples. This technique also allows the quantification of the expression of targeted miRNAs to validate observations determined by whole-genome screening or to analyze few specific miRNAs on a large number of samples. Here, we describe the validation of a method published by ABIs on their web site allowing to reverse transcribe and pre-amplify multiple miRNAs and snoRNAs simultaneously. The validation of this protocol was performed on human muscle and plasma samples. Fast and cost efficient, this method achieves an easy and convenient way to screen a relatively large number of miRNAs in parallel.

Development of a miRNA biochip platform

MicroRNAs (miRNAs) are short noncoding RNA molecules involved in the regulation of carcinogenesis. Tumor-derived miRNAs will be released into the circulation, and these miRNAs, also known as circulating miRNAs, have stable properties in plasma and serum. Thus miRNAs have been suggested as a potential biomarker for cancer diagnosis. The aim of this study was to develop a miRNA biochip platform. First, we used three types of reverse transcription (RT) primers; we constructed the miRNA-containing poly(A) tail and used RT primer oligo(dT) to make the complementary DNA (cDNA), and we also designed a linear-form RT primer and a stem-loop RT primer to synthesize cDNA. Second, we selected 11 candidate miRNAs related to colorectal cancer (CRC) from our previous studies and then designed the probes based on the sequence of the candidate miRNAs and constructed a miRNA biochip. miRNAs were extracted from CRC cell line SW620 and were detected by enzymatic biochip assay. According to the results, we found that this miRNA biochip can effectively detect the miRNAs by using a specific stem-loop RT primer to synthesize cDNA. In the future, we will design more oligonucleotides for miRNA detection and also evaluate the consistency between the results of the miRNA biochip and quantitative real-time polymerase chain reaction (PCR).

The allosteric HIV-1 integrase inhibitor BI-D affects virion maturation but does not influence packaging of a functional RNA genome.

The viral integrase (IN) is an essential protein for HIV-1 replication. IN inserts the viral dsDNA into the host chromosome, thereby aided by the cellular co-factor LEDGF/p75. Recently a new class of integrase inhibitors was described: allosteric IN inhibitors (ALLINIs). Although designed to interfere with the IN-LEDGF/p75 interaction to block HIV DNA integration during the early phase of HIV-1 replication, the major impact was surprisingly found on the process of virus maturation during the late phase, causing a reverse transcription defect upon infection of target cells. Virus particles produced in the presence of an ALLINI are misformed with the ribonucleoprotein located outside the virus core. Virus assembly and maturation are highly orchestrated and regulated processes in which several viral proteins and RNA molecules closely interact. It is therefore of interest to study whether ALLINIs have unpredicted pleiotropic effects on these RNA-related processes. We confirm that the ALLINI BI-D inhibits virus replication and that the produced virus is non-infectious. Furthermore, we show that the wild-type level of HIV-1 genomic RNA is packaged in virions and these genomes are in a dimeric state. The tRNAlys3 primer for reverse transcription was properly placed on this genomic RNA and could be extended ex vivo. In addition, the packaged reverse transcriptase enzyme was fully active when extracted from virions. As the RNA and enzyme components for reverse transcription are properly present in virions produced in the presence of BI-D, the inhibition of reverse transcription is likely to reflect the mislocalization of the components in the aberrant virus particle.

Deep sequencing of norovirus genomes defines evolutionary patterns in an urban tropical setting.

ABSTRACTNorovirus is a highly transmissible infectious agent that causes epidemic gastroenteritis in susceptible children and adults. Norovirus infections can be severe and can be initiated from an exceptionally small number of viral particles. Detailed genome sequence data are useful for tracking norovirus transmission and evolution. To address this need, we have developed a whole-genome deep-sequencing method that generates entire genome sequences from small amounts of clinical specimens. This novel approach employs an algorithm for reverse transcription and PCR amplification primer design using all of the publically available norovirus sequence data. Deep sequencing and de novo assembly were used to generate norovirus genomes from a large set of diarrheal patients attending three hospitals in Ho Chi Minh City, Vietnam, over a 2.5-year period. Positive-selection analysis and direct examination of protein changes in the virus over time identified codons in the regions encoding proteins VP1, p48 (NS1-2), and p22 (NS4) under positive selection and expands the known targets of norovirus evolutionary pressure.IMPORTANCE The high transmissibility and rapid evolutionary rate of norovirus, combined with a short-lived host immune responses, are thought to be the reasons why the virus causes the majority of pediatric viral diarrhea cases. The evolutionary patterns of this RNA virus have been described in detail for only a portion of the virus genome and never for a virus from a detailed urban tropical setting. We provide a detailed sequence description of the noroviruses circulating in three Ho Chi Minh City hospitals over a 2.5-year period. This study identified patterns of virus change in known sites of host immune response and identified three additional regions of the virus genome under selection that were not previously recognized. In addition, the method described here provides a robust full-genome sequencing platform for community-based virus surveillance.

The role of A-kinase anchoring protein 95-like protein in annealing of tRNALys3 to HIV-1 RNA.

BackgroundRNA helicase A (RHA), a DExH box protein, promotes annealing of tRNALys3, a primer for reverse transcription, to HIV-1 RNA and assembles into virus particles. A-kinase anchoring protein 95-like protein (HAP95) is a binding partner of RHA. The role of HAP95 in the annealing of tRNALys3 was examined in this study.ResultsHAP95 associates with the reverse transcriptase region of Pol protein of HIV-1. Decreasing endogenous HAP95 in HIV-1-producing 293T cells by siRNA reduces the amount of tRNALys3 annealed on viral RNA. This defect was further deteriorated by knockdown of RHA in the same cells, suggesting a cooperative effect between these two proteins. Biochemical assay in vitro using purified GST-tagged HAP95 shows that HAP95 may inhibit the activity of RHA.ConclusionThe results support a hypothesis that HAP95 may transiently block RHA’s activity to protect the annealed tRNALys3 on viral RNA in the cells from removing by RHA during the packaging of RHA into virus particles, thus facilitating the annealing of tRNALys3 to HIV-1 RNA.

Establishment and evaluation of direct assay for plasma microRNAs without RNA extraction by quantitative PCR

Objective To establish a new method for the detection of plasma miRNAs by real-time fluorescence quantitative PCR without RNA extraction. Methods 7.5 μl plasma separated from normal human blood was treated with equal amount of buffer containing Tween 20, which was used directly as sample for the following reverse transcription and PCR reaction to establish a direct assay for plasma microRNAs. U6 snRNA, miR-24-1-5p and miR-4634 were selected as target microRNAs for the determination of RT-qPCR. Both specificity and amplification efficiency were assessed. The difference of Cq values between traditional quantitative PCR detection method and our new method was also observed. Furthermore, the results of mixed primer or individual primer by RT-qPCR were compared in new established method. Results The amplification products of U6, miR-24-1-5p and miR-4634 show a single melting peak at 81 ℃, 83 ℃ and 84.2 ℃, respectively, without primer dimer peak or non-specific peak in all 8 cases of healthy human plasma samples. Amplification efficiency for miR-24-1-5p reached 1.1 detected in plasma direct assay. The amplification curve of U6, miR-24-1-5p and miR-4634 is parallel in each sample, which demonstrated that the amplification of slope is consistent. The Cq value of miR-4634 determined by direct assay is higher than the result of the method by RNA extraction（plasma direct assay 30.98±0.17，RNA extraction 29.22±0.21；t=8.475，P 0.05）and miR-24-1-5p(plasma direct assay 20.73±0.14，RNA extraction 21.06±0.21；t=1.749, P>0.05) although they had significant correlation（r=0.860 5,0.728 9,0.748 5,P<0.05）. No significant difference of Cq value between mixed primer reverse transcription and individual primer reverse transcription. Conclusion A novel RT-qPCR direct assay of plasma miRNAs is established, which can improve the efficiency of plasma miRNAs determination in potential clinical applications. （Chin J Lab Med，2014,37：119-122） Key words: MicroRNAs; Real-time polymerase chain reaction; Plasma

Circulating microRNAs: Understanding the Limits for Quantitative Measurement by Real‐Time PCR

Ever since the discovery of small, non-coding RNAs in circulation, microRNAs have gained significant attention as biomarkers or, in some instances, regulators of disease and/or associated complications. Until now, most articles published use real-time quantitative PCR (qPCR) for assessment of