RT Reaction Research Articles

Identification and optimization of parameters for accurate quantification of RNA by RT-dPCR.

Reverse transcription-digital PCR (RT-dPCR) is attracting attention as a method that enables SI-traceable RNA quantification without calibration, but its accuracy and bias have not been thoroughly studied. In this study, the accurate quantification of RNA by the RT-dPCR method was investigated using NMIJ CRM 6204-b, an RNA certified reference material whose certified value was assigned by orthogonal chemical measurement methods. Moreover, a two-step RT-dPCR method was adopted to examine in detail the conditions for the RT reaction process, which was expected to be the major uncertainty component in the RT-dPCR measurement. Optimization experiments revealed that the type of reverse transcriptase, the concentration of template RNA, and the type and concentration of primers in the RT reaction affected the value quantifiedby RT-dPCR. Under the optimal conditions, the value quantifiedby RT-dPCR, 76.4ng/μL ± 6.7ng/μL (the quantified value ± expanded uncertainty (k = 2)), was consistent with the certified value, 68.2ng/μL ± 5.8ng/μL, of NMIJ CRM 6204-b RNA 1000-A within the expanded uncertainty. From the results of the uncertainty evaluation, the relative combined uncertainty of the RT-dPCR method was 4.42%, and the major uncertainty components in the RT-dPCR method were the preparation of RT solution (3.68%), the inter-day difference (1.80%), and the RT reaction (1.30%). Together, the results suggested that the contribution of the RT reaction process to the total uncertainty was greater than that of the dPCR process.

Blocking Abundant RNA Transcripts by High-Affinity Oligonucleotides during Transcriptome Library Preparation

BackgroundRNA sequencing has become the gold standard for transcriptome analysis but has an inherent limitation of challenging quantification of low-abundant transcripts. In contrast to microarray technology, RNA sequencing reads are proportionally divided in function of transcript abundance. Therefore, low-abundant RNAs compete against highly abundant - and sometimes non-informative - RNA species.ResultsWe developed an easy-to-use strategy based on high-affinity RNA-binding oligonucleotides to block reverse transcription and PCR amplification of specific RNA transcripts, thereby substantially reducing their abundance in the final sequencing library. To demonstrate the broad application potential of our method, we applied it to different transcripts and library preparation strategies, including YRNAs in small RNA sequencing of human blood plasma, mitochondrial rRNAs in both 3′ end sequencing and long-read sequencing, and MALAT1 in single-cell 3′ end sequencing. We demonstrate that the blocking strategy is highly efficient, reproducible, specific, and generally results in better transcriptome coverage and complexity.ConclusionOur method does not require modifications of the library preparation procedure apart from simply adding blocking oligonucleotides to the RT reaction and can thus be easily integrated into virtually any RNA sequencing library preparation protocol.

Blood and Saliva Identification through miRNA in Forensic Molecular Biology

A class of non-coding miRNAs has attracted a lot of interest in the field of forensic sciences. miRNA has a relatively small size thus it is quite stable to the external environmental pressures and factors, which makes it very useful in forensic examination when used as a bio marker. At present, many of the specific miRNAs of body fluids can be distinguished by measuring their level of expression. Fresh blood samples and saliva samples were taken from 3 males and 3 females. RNA was extracted using TRIzol method and cDNA synthesis was done. RT reactions were made using SYBR Green master mix for RT-qPCR for quantification and detection of miRNAs. The designed primers were runs against the sample in qPCR and result was seen and compiled. Two miRNA were detected and expressed in both blood and saliva but they failed to differentiate between blood and saliva due to the non-specific primer binding according the methodology used in this study. Hsa-miR20a and Hsa-miR583 have been found to be non-specific for the blood and saliva. No stark difference could be observed on the basis of which we can say that Hsa-miR20a and HsamiR583 were specific for blood and saliva due to non-specific primer binding. Further research work is required in this domain of body fluids identification and differentiation using miRNA markers.

Development of a method for evaluating the mRNA transcription activity of influenza virus RNA-dependent RNA polymerase through real-time reverse transcription polymerase chain reaction

BackgroundThe development of an influenza RNA-dependent RNA polymerase (RdRp) inhibitor is required; therefore, a method for evaluating the activity of influenza RdRp needs to be developed. The current method uses an ultracentrifuge to separate viral particles and quantifies RdRp activity with radioisotope-labeled nucleosides, such as 32P-GTP. This method requires special equipment and radioisotope management, so it cannot be implemented in all institutions. We have developed a method to evaluate the mRNA transcription activity of RdRp without using ultracentrifugation and radioisotopes.ResultsRdRp was extracted from viral particles that were purified from the culture supernatant using anionic polymer-coated magnetic beads that can concentrate influenza virus particles from the culture supernatant in approximately 30 min. A strand-specific real-time reverse transcription polymerase chain reaction (RT-PCR) method was developed based on reverse transcription using tagged primers. RT primers were designed to bind to a sequence near the 3' end of mRNA containing a poly A tail for specific recognition of the mRNA, with an 18-nucleotide tag attached to the 5' end of the sequence. The RT reaction was performed with this tagged RT primer, and the amount of mRNA was analyzed using real-time qPCR. Real-time qPCR using the tag sequence as the forward primer and a segment-specific reverse primer ensured the specificity for quantifying the mRNA of segments 1, 4, and 5. The temperature, reaction time, and Mg2+ concentration were determined to select the optimum conditions for in vitro RNA synthesis by RdRp, and the amount of synthesized mRNAs of segments 1, 4, and 5 was determined with a detection sensitivity of 10 copies/reaction. In addition, mRNA synthesis was inhibited by ribavirin triphosphate, an RdRp inhibitor, thus indicating the usefulness of this evaluation method for screening RdRp inhibitors.ConclusionThis method makes it possible to analyze the RdRp activity even in a laboratory where ultracentrifugation and radioisotopes cannot be used. This novel method for measuring influenza virus polymerase activity will further promote research to identify compounds that inhibit viral mRNA transcription activity of RdRp.

Reference gene selection for miRNA and mRNA normalization in potato in response to potato virus Y

This was the first report on evaluating candidate reference genes for quantifying the expression profiles of both coding (e.g., mRNA) and non-coding (e.g., miRNA) genes in potato response to potato virus Y (PVY) inoculation. The reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) method was employed to quantify the expression profiles of eight selected candidate reference genes; their expression stability was analyzed by four statistical algorithms, i.e., geNorm, BestKeeper, NormFinder and RefFinder. The most stable reference genes were sEF1a, sTUBb and seIF5 with a high stability. The least stable ones were sPP2A, sSUI1 and sGAPDH. The same reference gene allows for normalization of both miRNA and mRNA levels from a single RNA sample using cDNAs synthesized in a single RT reaction, in which a stem-loop primer was used for miRNAs and the oligo (dT) for mRNAs.

Strand-specific detection of overlapping transcripts via purification involving denaturation of biotinylated cDNA.

Reverse transcription-PCR (RT-PCR) is the most widely employed technique for gene expression analysis owing to its high sensitivity, easy reproducibility and fast output. It has been conceived that priming RT reactions with gene-specific primers generates cDNA only from the specific RNA. However, several reports have revealed that cDNA is synthesized even without addition of exogenous primers in RT reactions. Owing to such self-priming activity, the signals from specific strands cannot be accurately detected and can confound the expression analysis, especially in context of overlapping bidirectional transcripts. Here, we demonstrate that purification of biotin-tagged cDNA in conjunction with alkaline denaturation can obviate the problem of background priming and enable accurate strand-specific detection of overlapping transcripts.

Detection of Human Immunodeficiency Virus Type 1 (HIV-1) Antisense Protein (ASP) RNA Transcripts in Patients by Strand-Specific RT-PCR.

In retroviruses, antisense transcription has been described in both human immunodeficiency virus type 1 (HIV-1) and human T-lymphotropic virus 1 (HTLV-1). In HIV-1, the antisense protein ASP gene is located on the negative strand of env, in the reading frame -2, spanning the junction gp120/gp41. In the sense orientation, the 3' end of the ASP open reading frame overlaps with gp120 hypervariable regions V4 and V5. The study of ASP RNA has been thwarted by a phenomenon known as RT-self-priming, whereby RNA secondary structures have the ability to prime RT in absence of the specific primer, generating non-specific cDNAs. The combined use of high RNA denaturation with biotinylated reverse primers in the RT reaction, together with affinity purification of the cDNA onto streptavidin-coated magnetic beads, has allowed us to selectively amplify ASP RNA in CD4+ T cells derived from individuals infected with HIV-1. Our method is relatively low-cost, simple to perform, highly reliable, and easily reproducible. In this respect, it represents a powerful tool for the study of antisense transcription not only in HIV-1 but also in other biological systems.

A Comparison of Two Standardized Quantitative RT-PCRs with CE IVD Kit for Digital PCR in CML Patients with Different Level of BCR-ABL1 Transcripts

The detection and quantification of BCR-ABL1 transcripts play a key role in therapy management of chronic myeloid leukemia (CML) patients treated with tyrosine kinase inhibitors (TKIs). Reverse transcription quantitative PCR (RT-qPCR) is considered the gold standard strategy for monitoring of minimal residual disease (MRD). Recently, digital PCR (dPCR) was introduced as a new quantification method based on determination of absolute target sequence quantity. In connection with the deep molecular response (MR) assessment and the option of TKIs treatment cessation, the dPCR has a potential of high sensitivity, robustness and accuracy of BCR-ABL1 transcripts detection. The aim of present work was to compare the results of two standardized RT-qPCR methods with RT-dPCR detection in clinical samples of CML patients with different BCR-ABL1 fusion transcripts level. In total, 62 peripheral blood samples of CML patients with the level of BCR-ABL1 transcripts (international scale, IS) between 100% and undetectable were enrolled. The samples were analyzed by three methods: (1) CE IVD Xpert BCR-ABL Ultra Kit (GeneXpert, Cepheid), (2) conventional RT-qPCR assay according to ELN guidelines and certified by EUTOS for the level of MR4.5 BCR-ABL1 detection (ABI7300, Applied Biosystems), and (3) RT-dPCR using CE IVD QXDx BCR-ABL %ID Kit (Bio-Rad) on QX200 Droplet Digital PCR System (Bio-Rad). All steps were performed according to manufacturer´s instructions. A linear regression analysis and an overall reporting bias analysis using the Bland-Altman test were used for comparison of the methods. Based on the results of GeneXpert as the determined routine method for BCR-ABL1 transcripts quantification, the samples were divided into two groups: 50/62 samples were scored as BCR-ABL1 positive (the first group), while 12/62 samples were scored as BCR-ABL1 undetectable (the second group). The first group of GeneXpert positive samples was analyzed using both conventional RT-qPCR and RT-dPCR (both in duplicates). The median of the sum of ABL copies per sample was 180,511 copies (range 56,466 - 536,453) by RT-qPCR vs. 39,960 (range 14,360 - 71,690) by RT-dPCR. Both conventional RT-qPCR and RT-dPCR did not detect any BCR-ABL1 transcript in 4/50 samples. In addition, RT-dPCR did not detect any BCR-ABL1 transcript in 5 other samples (Figure 1). Linear regression analysis showed good correlation between both sets of assays: GeneXpert vs. RT-dPCR (R2=0.965, N=41) and conventional RT-qPCR vs. RT-dPCR (R2=0.977, N=41). The slopes of regression curve were not significantly different from the value of 1 for both sets of the compared assays (1.02; 95% CI, range 0.95 - 1.08 and 1.02; 95% CI, range 0.97 - 1.08, respectively). In addition, an overall bias (Bland-Altman analyses) was -0.05 (GeneXpert vs. RT-dPCR) and -0.12 (conventional RT-qPCR vs. RT-dPCR) suggesting good concordance in % IS reporting between RT-dPCR and two standardized quantitative BCR-ABL1 assays. The evaluation of MR level was identical by all three methods in 26/50 samples. The comparison of two methods revealed consistent MR level by GeneXpert vs. RT-dPCR in 29/50 samples and by conventional RT-qPCR vs. RT-dPCR in 28/50 samples. The RT-dPCR scored 12/50 samples (GeneXpert vs. RT-dPCR) and 13/50 samples (conventional RT-qPCR vs. RT-qPCR) to the different level of MR compared to standardized RT-qPCR methods. The second group of samples (12/62) undetectable for BCR-ABL1 transcripts according to GeneXpert was analyzed using conventional RT-qPCR and RT-dPCR in tetraplicates to reach higher sensitivity. The BCR-ABL1 negativity was confirmed in 11/12 samples. One sample was BCR-ABL1 positive by both the conventional RT-qPCR (0.0008% IS) and RT-dPCR (0.0013% IS). We showed that the results of quantitative detection of BCR-ABL1 transcripts (% IS) obtained by the CE IVD RT-dPCR kit are in good correlation with the results of both standardized RT-qPCR methods and all three methods provide comparable sensitivity. RT-qPCR method yielded higher number of copies of control ABL gene per sample compared to RT-dPCR although the input amount of RNA into the RT reaction was the same. The MR level evaluation of RT-dPCR vs. both RT-qPCR methods was identical to the level MR3.0 (category >0.01% IS), while for samples in deep MR (category ≤ 0.01% IS, MR4.0 and below) revealed partial difference in categorization into the individual MR levels. Supported by MH CZ - DRO (FNBr, 65269705). Disclosures Zackova: Novartis: Consultancy; Bristol Myers Squibb: Consultancy; Angelini: Consultancy; Incyte: Consultancy. Mayer:AOP Orphan Pharmaceuticals AG: Research Funding.

Flèche versus Lunge as the Optimal Footwork Technique in Fencing.

The objective of the study reported in this paper involved identifying the fencing attack (flèche versus lunge) that provides greater effectiveness in a real competition. Two hypotheses are presented in the study. The first hypothesis involves the greater effectiveness of the flèche with regard to bioelectric muscular tension, and the second hypothesis involves the reduction of movement time of the flèche. Therefore, analyses were conducted by the application of EMG (electromyography) signal, ground reaction forces, and parameters representing sensorimotor responses (RT—reaction time and MT—movement time). This study included six world-leading female épée fencers (mean age: 24.6 ± 6.2 years). Throughout the procedure, the subjects performed flèche and lunge touches at the command of the coach based on visual stimuli. The experimental results indicated the greater effectiveness of the flèche compared with the lunge with regard to increases in EMG values (p = 0.027) in the lateral and medial gastrocnemius muscles and decreases in the duration of the movement phase (p = 0.049) and vertical force of the rear leg (p = 0.028). In conclusion, higher levels of EMG and ground reaction forces were generated during the flèche compared with the lunge, which promotes an improvement in the explosive force and contributes to a reduction in the movement phase of the entire offensive action.

Chromium 10× Single-Cell 3' mRNA Sequencing of Tumor-Infiltrating Lymphocytes.

Chromium 10× 3' V2 protocol is a 3' end counting single-cell mRNA sequencing protocol that allows to process and sequence RNA from thousands of cells in parallel. Chromium10× by 10× Genomics is an emulsion-based device that enables to compartmentalize single cells along with sets of uniquely barcoded primers and reverse transcription reagents into nanoscale droplets that are used as reaction chambers to generate barcoded full-length cDNA from single cells. After RT reaction single-stranded barcoded cDNAs are pooled together and processed to generate sequencing libraries compatible with the standard Illumina platforms. Here we show in detail the main steps of the protocol applied to the analysis of tumor-infiltrating T lymphocytes (TILs). The main steps are cell preparation, cDNA synthesis, library construction, and sequencing.This protocol refers specifically to the CG00052_SingleCell3_ReagentKitv2UserGuide_RevD downloadable from 10× Genomics website ( https://www.10xgenomics.com ) and does not substitute it. Always refer to this guide, paying attention to updates and revisions.

Iodo-Cycloisomerization of Aryl(indol-3-yl)methane-Tethered Propargyl Alcohols to 3-Iodocarbazoles via Selective 1,2-Alkyl Migration.

Herein, we disclose the first report on iodo-cycloisomerization of 1-(indol-3-yl)-1-arylbut-3-yn-2-ols to form 3-iodocarbazoles. The synthesis proceeds through a cascade 5-endo-spirocyclization, followed by selective 1,2-alkyl migration. This method governs the green synthesis principles such as open-flask reaction, AcOEt as the solvent, rt reaction with short time, use of iodine, and broad substrate scope with atom and step economy.

The laterality of stop and go processes of the motor response in left-handed and right-handed individuals

ABSTRACTThe objective of the present study was to investigate whether the stop and go processes of the motor response are asymmetrical and whether the asymmetries are dependent on handedness and the response selection process that is engaged. Both right-handed and left-handed participants abducted either the left or right index finger in response to an imperative cue in the choice reaction time (choice RT) or the simple RT task. A stop cue was presented after the imperative cue with a probability of .25. When the stop cue was presented, the participants withheld the prepared response. On the choice RT task, left-handed participants had significantly shorter RT and stop signal reaction time (SSRT) with the left versus the right hand, whereas right-handers showed no difference between hands on either measure. In the simple RT task, the RT and SSRT were not significantly different between the groups or the response sides. These results indicate that both the stop and go processes of the prepared left-hand response are completed earlier than those of the right-hand response in left-handed individuals when the stimulus-response process involves a response selection process.

Variables influencing the efficiency and interpretation of reverse transcription quantitative PCR (RT-qPCR): An empirical study using Bacteriophage MS2

Reverse transcription, quantitative PCR (RT-qPCR) is a sensitive method for quantification of specific RNA targets, but the first step of the assay, reverse transcription, is notoriously variable and sensitive to reaction conditions. In this study, we used purified Bacteriophage MS2 genomic RNA as a model virus target to test two different RT enzymes (SuperScript II and SuperScript III), two RT-priming strategies (gene-specific primers and random hexamers), and varying background RNA concentrations (0–50ngμl−1) to determine how these variables influence the efficiency of reverse transcription over a range of target concentrations (101–107 copies μl−1). The efficiency of the RT reaction was greatly improved by increasing both background RNA and primer concentrations, but the benefit provided by background RNA was source dependent. At a given target concentration, similar RT efficiencies were achieved with gene-specific primers and random hexamers, but the latter required much higher concentrations. With random hexamers, we observed a systematic variation in RT reaction efficiency as a function of target concentration. Using an RNA standard curve that was also subject to RT effectively normalized for this systematic variability, but the assay accuracy depended critically on the length of the standard RNA extending to the 3' end of the qPCR target site. Our results shed some light on previous contradictory conclusions in the literature, and provide insights that may aid in the design of RT-qPCR assays and the design of synthetic RNA standards when full-length material is not available.

Profiling Circulating miRNAs from the Plasma of Individuals with Metabolic Syndrome.

The technique of RT-qPCR (real time-quantitative polymerase chain reaction) is invaluable in miRNA research both at the profiling and individual RT-qPCR stages. At the profiling stage, numerous miRNAs are looked at in the plasma of numerous individuals from two or more cohorts (i.e., control vs. case). The miRNAs of interest would be either upregulated or downregulated by more than twofold in the case cohort compared to the control cohort. Profiling human specimens for miRNA biomarkers has exploded over the last decade, with researchers profiling plasma, serum, urine, and also the miRNA content of extracellular vesicles, which are also isolated from human specimens. RT-qPCR is a relatively easy technique; however, sample preparation from plasma to RNA to RNA input in RT reaction requires accuracy and precision.

First Report of Broad bean wilt virus 2 in Dioscorea opposita Thunb. in Korea

HomePlant DiseaseVol. 100, No. 2First Report of Broad bean wilt virus 2 in Dioscorea opposita Thunb. in Korea PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of Broad bean wilt virus 2 in Dioscorea opposita Thunb. in KoreaS.-J. Kwon, I.-S. Cho, J.-Y. Yoon, S.-K. Choi, and G.-S. ChoiS.-J. KwonSearch for more papers by this author, I.-S. ChoSearch for more papers by this author, J.-Y. YoonSearch for more papers by this author, S.-K. ChoiSearch for more papers by this author, and G.-S. ChoiSearch for more papers by this authorAffiliationsAuthors and Affiliations S.-J. Kwon I.-S. Cho J.-Y. Yoon S.-K. Choi G.-S. Choi , Horticultural and Herbal Crop Environment Division, National Institute of Horticultural and Herbal Science, RDA, Wanju 565-862, Republic of Korea. Published Online:4 Dec 2015https://doi.org/10.1094/PDIS-07-15-0752-PDNAboutSectionsSupplemental ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat Broad bean wilt virus 2 (BBWV2), a member of the genus Fabavirus, infects a wide range of plants, most of which are important agricultural and horticultural crops. In May 2015, to examine BBWV2 incidence in Chinese yam fields in Korea, two open fields were randomly selected in Andong City where Chinese yam has been cultivated intensively. Chinese yam plants showing virus-like symptoms, such as mosaic, stunting, mottle, yellowing, and leaf distortion, were easily found at a relatively high incidence of 10 to 15% in the surveyed fields. A total of 40 leaf samples were collected and evaluated for the presence of BBWV2 using a commercial double-antibody sandwich (DAS)-ELISA kit that detect both Broad bean wilt virus 1 (BBWV1) and BBWV2 (Agdia, Elkhardt, IN, USA). Interestingly, all the collected samples were positive by DAS-ELISA. To confirm the result from the DAS-ELISA assay and to specify whether the samples were infected with either BBWV1 or BBWV2, reverse transcription-polymerase chain reaction (RT-PCR) was performed using two sets of the primers that amplify specifically BBWV1 (5′-TGACACATATGTGGCCATGG-3′ and 5′-CAGATTCTCAAGTTGGTGACAGG-3′) and BBWV2 (5′-CAGAGTTCAGTAGTTCCTGCTTATG-3′ and 5′-GGCATTTCAACCCTGCATAATAC-3′) as described previously (Panno et al. 2014). The RT-PCR result showed that all the samples were infected with BBWV2 and no amplification was detected when using the BBWV1-specific primer set. One of the samples infected with BBWV2 was selected to determine complete genome sequence of BBWV2 isolated from Chinese yam in Korea. Total RNA extracted from the sample infected with BBWV2 was subjected to RT reactions using the Superscript III reverse transcription (Invitrogen, Carlsbad, CA, USA) and either the primer BBWVR1-3E-Rv (5′-CCCTCACTACTGAAATTTACTT-3′, designed to hybridize to the 3′ terminus of BBWV2 RNA1), or the primer BBWVR2-3E-Rv (5′-AAAATACTACTAAAGCTTATACATATA-3′, designed to hybridize to the 3′ terminus of BBWV2 RNA2). Full-length cDNAs of BBWV RNA1 and RNA2 were amplified by PCR using Phusion high-fidelity DNA polymerase (NEB, Beverly, MA, USA) with the primer BBWV-5E-Fw (5′-GTTTTAATATTTTATTAAAACAAACAGCT-3′, designed to hybridize to the 5′ terminus of BBWV2 RNA1 and RNA2) and either the primer BBWVR1-3E-Rv or BBWVR2-3E-Rv, respectively. Sequencing of the BBWV genome and the 5′ and 3′ rapid amplification of cDNA ends (RACE) of BBWV RNAs were performed as described previously (Kwon et al. 2014). The assembled full-length sequences of RNA1 and RNA2 of BBWV2 isolated from Chinese yam were 5954 and 3598 nucleotides in length, respectively. The sequences were deposited in GenBank under the Accession Nos. KT246495 and KT246496, respectively. Nucleotide blast searches showed that RNA1 and RNA2 of the BBWV2 isolate from Chinese yam have maximum nucleotide identities of 95% to RNA1 (Accession No. KJ789136) and 95% to RNA2 (KJ789137) of the isolate SG1 of BBWV2, which was isolated from D. opposite in China. To the best of our knowledge, this is the first report of BBWV2 in Chinese yam in Korea. As a high incidence of BBWV2 in Chinese yam was found in this study, the effect of BBWV2 infection on yields of Chinese yam production needs to be evaluated by further studies. Our results also suggest that development of management practices for BBWV2 and production system for virus-free seedling is required to increase Chinese yam yield in Korea.

β2-Microglobulin is an appropriate reference gene for RT-PCR-based gene expression analysis of hematopoietic stem cells.

Real-time reverse transcription polymerase chain reaction (RT-PCR) is regarded as one of the most useful and powerful tools for characterizing hematopoietic stem cells (HSCs), because samples of extremely small cell numbers can be analyzed. The expression levels determined by RT-PCR are based on relative quantification; therefore, the selection of an appropriate reference gene with a relatively stable expression level under most conditions is crucial. Here, we determined that beta2-microglobulin (B2m) is an appropriate reference gene for analyzing mouse HSCs by a novel method using single-cell RT-PCR. Clonally sorted HSCs were subjected to RT reactions with exogenous RNA fragments and then to real-time PCR. Next, the relative gene expression levels of 4 well-known housekeeping genes were quantified in each single cell sample based on the threshold cycle of exogenous RNA. The analysis revealed that B2m expression was reproducibly detected in almost all HSCs and that B2m had the most stable expression level among the compared genes, even after the cells had been cultured under various conditions. Thus, our results indicate that B2m can reliably be used as a reference gene for the relative quantification of expression levels in HSCs across various conditions. Furthermore, our work proposes a novel method for the selection of appropriate reference genes.

Self-priming on the plant viral RNAs during reverse transcription-PCR.

The occurrence of the primer-independent cDNA synthesis during RT-PCR analysis of human and animal RNA viruses has been well documented. Conversely, there is scant knowledge about this event in plant RNA viruses. Here we show that the primer-independent cDNA synthesis occurs in all eight different plant RNA viruses tested in this study, suggesting a common phenomenon for RT-PCR analysis of plant RNA viruses. Additional experiments indicate that the event is likely contributed to by RNA self-priming, and can be effectively reduced or eliminated through increasing temperature of the RT reaction.

Abstract 5233: MicroRNA in biofluid as robust biomarkers for cancer

Abstract microRNAs constitute a class of small cellular RNAs (typically 19-23 nt) that function as post-transcriptional regulators of gene expression. Current estimates indicate that more than one third of the human cellular transcriptome is regulated by this small class of RNA (∼2000 miRNA). The study of extracellular microRNAs and their potential as pathophysiological markers has greatly expanded in the last couple of years. microRNAs have been shown to be actively exported from tissues into the circulation through a variety of mechanisms including exosome and microvesicle transport, and complexing with RNA binding proteins or HDL. The high relative stability of microRNAs in common clinical source materials (FFPE blocks, plasma, serum, urine, saliva, etc.) and the ability of microRNA expression profiles to accurately classify discrete tissue types and specific disease states have positioned microRNAs as promising new biomarkers for diagnostic application in cancer. We have applied Exiqons highly sensitive LNA™-based qPCR platform for detection of microRNAs, which has enabled microRNA profiling in biofluids where levels are extremely low. The platform uses a single RT reaction to conduct full miRNome profiling and allows high-throughput profiling of microRNAs without the need for pre-amplification. Thousands of biofluid samples including serum/plasma and urine have been profiled to determine normal reference ranges for circulating microRNAs as well as to identify biomarkers of disease. Extensive data qualification and analysis methods have been developed and are central parameters to secure high quality data from biofluids. The methods can quickly and robustly be applied in biomarker discovery and validation projects. We will present examples from our collaborative cancer diagnostic projects. Also we have developed a new exosome enrichment method and will present a comparison of microRNA profiles obtained with this method to profiles obtained with different commercial available exosome isolation methods and standard profiles of whole plasma and serum. Citation Format: Thorarinn Blondal, Anni Thomsen, Jörg Krummheyer, Peter Mouritzen, Ditte Andreasen, Maria Theilum, Jan Stenvang, Claus L. Andersen, Hans J. Nielsen, Nils Brünner. MicroRNA in biofluid as robust biomarkers for cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5233. doi:10.1158/1538-7445.AM2014-5233

Placental Leucine Aminopeptidase/Oxytocinase Expression in Miscarriage

Objectives: To evaluate the role of placental leucine aminopeptidase (P-LAP) in miscarriage and searching to select the gene of this enzyme in miscarriage to exclude direct or indirect effects of abortion. Methods: Total RNA is purified from the fresh placental tissue sample according to the protocol of the QIAamp®RNA Tissues Mini Kit (Qiagen). The sample is visualized by 0.7% agrose gel containing. Total RNA is reverse-transcribed RT-PCR carried out on the previously extracted RNA samples using (QIAGEN One Step RT –PCR Kit). Aliquots (1ml) of RT reaction samples are amplified by PCR, the PCR products (10μl) per lane are resolved by using electrophoresis on 2.0% agrose gel .Later the product is detected and examined under UV transiluminator. Results: The obtained results indicate that there is an expression of P-LAP mRNAs in placentas of normal pregnancy women (28±SE0.45) and there is a decrease in miscarriage. Furthermore, there is a difference between single miscarriage and those with recurrent miscarriage, the mean values in single miscarriage (1 and 2) are (16.1%±SE0.84) and (13%±SE0.24) respectively while the mean values in recurrent miscarriage (1 and 2) are (12%±SE0.19) and (9%±SE0.52) respectively. The expression of P-LAP is significantly lower (p=0.05) in miscarriage and highly significant decrease in recurrent than in single miscarriage women. Original Research Article British Journal of Medicine & Medical Research, 4(17): 3283-3292, 2014 3284 Conclusion: The decrease of P-LAP mRNA levels in miscarriage placentas could be a sign that p-LAP function is an important step in pregnancy regulation.

Development and application of a novel reverse transcription real-time PCR method for miR-499 quantification

ObjectivesMicroRNAs (miRNAs) are endogenous small RNAs of 21–25 nucleotides that can pair with sites in 3′ untranslated regions in mRNAs of protein-coding genes to downregulate their expression. Recently, miR-499 and other miRNAs released in circulating blood have been reported as promising biomarkers for acute myocardial infarction (AMI). In the present study, we developed a novel reverse-transcription real-time PCR assay for human miR-499 quantification. Design and methodsmiR-499 was reverse-transcribed with a 3′ portion-specific primer into cDNAs. The cDNAs were further extended with overlap PCR. The extended cDNAs were determined by quantitative, real-time PCR. Synthetic miR-499 was put into the RT reaction over an optimal range to generate standard curves for absolute quantification of miR-499. ResultsIn the presence of 0.0001amol/μL to 1.0×106amol/μL of synthetic miR-499, we observed a linear correlation (R2=0.999) between the logarithm of the amount of input RNA and the CT value. The miR-499 was reliably measured at a detection limit of 0.0001amol/μL. The miR-499 measurements in spiked plasma samples indicated excellent correlation between the novel qRT PCR and classic stem loop qRT PCR. The qRT PCR analysis demonstrated that miR-499 was detected with higher levels in plasma from the patient with AMI in acute phase (AMI) compared with those from the control groups (P<0.001). ConclusionsWe developed a novel reverse-transcription real-time PCR assay for human miR-499 quantification. The good reproducibility and wide linearity range may permit more use of it in the quantification of other human miRNAs in future.