Variability of the Reverse Transcription Step: Practical Implications.
Bustin SA, Dhillon HS, Kirvell S, Greenwood C, Parker M, Shipley GL, Nolan T.
Clin Chem. 2014 Oct 31

BACKGROUND: The reverse transcription (RT) of RNA to cDNA is a necessary first step for numerous research and molecular diagnostic applications. Although RT efficiency is known to be variable, little attention has been paid to the practical implications of that variability.
METHODS: We investigated the reproducibility of the RT step with commercial reverse transcriptases and RNA samples of variable quality and concentration. We quantified several mRNA targets with either singleplex SYBR Green I or dualplex probe-based real-time quantitative PCR (qPCR), with the latter used to calculate the correlation between quantification cycles (Cqs) of mRNA targets amplified in the same qPCR assay.
RESULTS: RT efficiency is enzyme, sample, RNA concentration, and assay dependent and can lead to variable correlation between mRNAs from the same sample. This translates into relative mRNA expression levels that generally vary between 2- and 3-fold, although higher levels are also observed.
CONCLUSIONS: Our study demonstrates that the variability of the RT step is sufficiently large to call into question the validity of many published data that rely on quantification of cDNA. Variability can be minimized by choosing an appropriate RTase and high concentrations of RNA and characterizing the variability of individual assays by use of multiple RT replicates.

Comparison of reverse transcriptases in gene expression analysis.
Stahlberg A, Kubista M, Pfaffl M.
Department of Chemistry and Biosciences, Chalmers University of Technology, Gothenburg, Sweden.
Clin Chem. 2004 Sep;50(9):1678-80.

Properties of the reverse transcription reaction in mRNA quantification.
Stahlberg A, Hakansson J, Xian X, Semb H, Kubista M.
Department of Chemistry and Bioscience, Chalmers University of Technology, Gothenburg, Sweden.
Clin Chem. 2004 Mar;50(3):509-15. Epub 2004 Jan 15


BACKGROUND: In most measurements of gene expression, mRNA is first reverse-transcribed into cDNA. We studied the reverse transcription reaction and its consequences for quantitative measurements of gene expression.
METHODS: We used SYBR green I-based quantitative real-time PCR (QPCR) to measure the properties of reverse transcription reaction for the beta-tubulin, glyceraldehyde-3-phosphate dehydrogenase, Glut2, CaV1D, and insulin II genes, using random hexamers, oligo(dT), and gene-specific reverse transcription primers.
RESULTS: Experimental variation in reverse transcription-QPCR (RT-QPCR) was mainly attributable to the reverse transcription step. Reverse transcription efficiency depended on priming strategy, and the dependence was different for the five genes studied. Reverse transcription yields also depended on total RNA
CONCLUSIONS: RT-QPCR gene expression measurements are comparable only when the same priming strategy and reaction conditions are used in all experiments and the samples contain the same total amount of RNA. Experimental accuracy is improved by running samples in (at least) duplicate starting with the reverse transcription reaction.

Low efficiency of the Moloney murine leukemia virus reverse transcriptase
during reverse transcription of rare t(8;21) fusion gene transcripts.

Curry J, McHale C, Smith MT.
Biotechniques. 2002 Apr;32(4):768, 770, 772, 754-5.
Department of Immunology, Molecular and Cellular Biology, University of California, Berkeley 94720-3200, USA.

The resolving power of RT-PCR is limited by the efficiency of RNA-to-cDNA conversion. Methods to determine this efficiency, using a real-time PCR assay for quantifying AML1-MTG 8 [t(8;21)] fusion gene transcripts, are described. The efficiency is shown to be directly proportional to RNA template levels. The Moloney murine leukemia virus (MMLV) reverse transcriptase enzyme's conversion efficiency was calculated to be approximately 20%. The efficiency was even lower (6%) when target templates were rare (single molecules) in the RT reactions. Levels of nonspecific or background RNA present in the RT reaction reduced the reverse transcriptase's conversion efficiency. This background effect was particularly pronounced when the specific template was present in rare amounts.

Increased Yield of PCR Products by Addition of T4 Gene 32 Protein
to the SMART PCR cDNA Synthesis System.

C. Villalva, C. Touriol, P. Seurat, P. Trempat, G. Delsol, and P. Brousset
Centre Hospitalier Universitaire de Purpan Toulouse, France
BioTechniques 31:81-86 (July 2001)

Under certain conditions, T4 gene 32 protein is known to increase the efficiency of different enzymes, such as Taq DNA polymerase, reverse transcriptase, and telomerase. In this study, we compared the efficiency of the SMART PCR cDNA synthesis kit with and without the T4 gene 32 protein. The use of this cDNA synthesis procedure, in combination with T4 gene 32 protein, increases the yield of RT-PCR products from approximately 90% to 150%. This effect is even observed for long mRNA templates and low concentrations of total RNA (25 ng). Therefore, we suggest the addition of T4 gene 32 protein in the RT-PCR mixture to increase the efficiency of cDNA synthesis, particularly in cases when low amounts of tissue are used.

Acoustic microstreaming increases the efficiency of reverse transcription reactions comprising single-cell quantities of RNA.
Boon WC, Petkovic-Duran K, White K, Tucker E, Albiston A, Manasseh R, Horne MK, Aumann TD.
Biotechniques. 2011 Feb;50(2):116-169
Florey Neuroscience Institutes, The University of Melbourne, Parkville, Victoria, Australia; Centre of Neuroscience, The University of Melbourne, Parkville, Victoria, Australia

Correlating gene expression with behavior at the single-cell level is difficult, largely because the small amount of available mRNA (<1 pg) degrades before it can be reverse transcribed into a more stable cDNA copy. This study tested the capacity for a novel acoustic microstreaming method ("micromixing"), which stirs fluid at microliter scales, to improve cDNA yields from reverse transcription (RT) reactions comprising single-cell quantities of RNA. Micromixing significantly decreased the number of qPCR cycles to detect cDNA representing mRNA for hypoxanthine phosphoribosyl-transferase (Hprt) and nuclear receptor-related 1 (Nurr1) by ~9 and ~15 cycles, respectively. The improvement was equivalent to performing RT with 10- to 100-fold more cDNA in the absence of micromixing. Micromixing enabled reliable detection of the otherwise undetectable, low-abundance transcript, Nurr1. It was most effective when RNA concentrations were low (0.1-1 pg/µL, a "single-cell equivalent") but had lesser effects at higher RNA concentrations (~1 ng/µL). This was supported by imaging experiments showing that micromixing improved mixing of a low concentration (20 pg/µL) of fluorescence-labeled RNA but not a higher concentration (1 ng/µL). We conclude that micromixing significantly increases RT yields obtainable from single-cell quantities of RNA.

Evaluation of sense-strand mRNA amplification by comparative quantitative PCR.
Loyal A. Goff1, Jessica Bowers2, Jaime Schwalm2, Kevin Howerton2, Robert C. Getts 2, and Ronald P. Hart1*
1W.M. Keck Center for Collaborative Neuroscience, Rutgers University, Piscataway, NJ 08854 USA
2Genisphere, Inc., Hatfield, PA 19440 USA
BMC Genomics 2004, 5:76

Background RNA amplification is required for incorporating laser-capture microdissection techniques into microarray assays. However, standard oligonucleotide microarrays contain sensestrand probes, so traditional T7 amplification schemes producing anti-sense RNA are not appropriate for hybridization when combined with conventional reverse transcription labeling methods. We wished to assess the accuracy of a new sense-strand RNA amplification method by comparing ratios between two samples using quantitative realtime PCR (qPCR), mimicking a two-color microarray assay.
Results We performed our validation using qPCR. Three samples of rat brain RNA and three samples of rat liver RNA were amplified using several kits (Ambion messageAmp, NuGen Ovation, and several versions of Genisphere SenseAmp). Results were assessed by comparing the liver/brain ratio for 192 mRNAs before and after amplification. In general, all kits produced strong correlations with unamplified RNAs. The SenseAmp kit produced the highest correlation, and was also able to amplify a partially degraded sample accurately.
Conclusion We have validated an optimized sense-strand RNA amplification method for use in comparative studies such as two-color microarrays.

Optimization of RNA yield, purity and mRNA copy number
by treatment of urine cell pellets with RNAlater.

M. Medeirosa,b, V.K. Sharmaa, R. Dinga, K. Yamajia, B. Lia, T. Muthukumara,
S. Valderde-Rosasb, A.M. Hernandezb, R. Mun˜ozb, M. Suthanthirana,*
aWeill Medical College of Cornell University, New York, NY, USA
b Hospital Infantil de Mexico, Federico Gomez, Mexico D.F., Mexico
Journal of Immunological Methods 279 (2003) 135– 142

Background: We have shown that measurement of mRNA for cytotoxic attack proteins perforin and granzyme B in urinary cells is a noninvasive means of diagnosing acute rejection of human renal allografts. Urinary cell mRNA studies have yielded useful information in other patient populations such as patients with cancer. The isolation of sufficient and high quality ribonucleic acid (RNA) from urinary cells however is problematic. RNAlater, an RNA stabilization solution, has been reported to optimize RNA isolation from tumor tissues stored at room temperature and from pigment-rich ocular tissues.
Methods: We
explored whether the addition of RNAlater to urine cell pellets improves RNA yield, enhances purity and facilitates measurement of low abundance mRNAs. We measured, with the use of real-time quantitative polymerase chain reaction (PCR) assay, levels of expression of a constitutively expressed gene 18S rRNA and mRNA for granzyme B and transforming growth factor-h1 (TGF-h1) in urine specimens and renal biopsies obtained from renal allograft recipients.
Results: RNAyield ( P < 0.01,
Wilcoxon signed rank test) and the A260/A280 ratio ( P < 0.01) were both higher with urine cell pellets treated with RNAlater prior to snap freezing compared to cell pellets that were not treated with RNAlater prior to snap freezing. Levels (copy number per 1 Ag of total RNA) of 18S rRNA ( P < 0.02), granzyme B mRNA ( P= 0.002) and TGF-h1 ( P= 0.02) were all higher with treated urine cell pellets compared to untreated cell pellets. Kruskall–Wallis one way analysis of variance and pair-wise comparisons with Student–Newman–Keuls test showed that the levels of mRNA for granzyme B ( P < 0.05) and TGF-h1 ( P < 0.05) are significantly different between renal allograft biopsies and untreated urine cell pellets but not between the biopsy specimens and RNAlater-treated urine cell pellets.
Conclusions: The addition of RNAlater to urine cell pellets improves RNA
isolation from urinary cells and facilitates measurement of low abundance mRNAs.

Global amplification of sense RNA: a novel method to replicate and
archive mRNA for gene expression analysis.

Mangalathu S. Rajeevan,a,* Irina M. Dimulescu,a Suzanne D. Vernon,a Mukesh Verma,b and Elizabeth R. Ungera
Viral Exanthems and Herpesvirus Branch, Division of Viral and Rickettsial Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, U.S. Department of Health and Human Services, Atlanta, GA 30333, USA
b Division of Cancer Prevention, National Cancer Institute, Bethesda, MD 20852, USA
Genomics 82 (2003) 491–497

We have developed a procedure to amplify mRNA into sense RNA (sRNA) so as to create a regenerating biorepository representing the complex mRNA profile in the original sample. The procedure exploits the template-switching activity of reverse transcriptase to incorporate RNA polymerase binding sites upstream of single-stranded cDNA (ss cDNA). Limited PCR was used for double-stranded DNA (dsDNA) synthesis. sRNA was synthesized from PCR products by in vitro transcription (IVT). sRNA was evaluated by real-time reverse transcription (RT)-PCR. sRNA synthesis was successful with RNA from human cell lines and tissues, yielding 2000- to 2500-fold amplification of glyceraldeyde-3 phosphate dehydrogenase (G3PDH). The size of sRNA ranged from 3.0 to 0.1 kb. sRNA synthesis preserved the relative differences in plant mRNAs spiked at abundance ranging over 5 orders of magnitude (0.00001– 0.1%). This reflects the high fidelity of sRNA synthesis for mRNA as low as 0.3 copies/cell. sRNA is amplified synthetic mRNA in the 533 direction; the appropriate template for any gene expression analysis.

RNA amplification strategies for cDNA microarray experiments.
Wang J, Hu L, Hamilton SR, Coombes KR, Zhang W.
University of Texas M.D. Anderson Cancer Center, Houston, TX, USA.
Biotechniques. 2003 Feb;34(2):394-400

The biological materials available for cDNA microarray studies are often
limiting. Thus, protocols have been developed to amplify RNAs isolated from limited amounts of tissues or cells. RNA amplification by in vitro transcription is the most widely used among the available amplification protocols. Two means of generating a dsDNA template for the RNA polymerase are a combination of reverse transcription with conventional second-strand cDNA synthesis and a combination of the switch mechanism at the 5' end of RNA templates (SMART) with reverse transcription, followed by PCR. To date, there has been no systematic comparison of the efficiency of the two amplification strategies. In this study, we performed and analyzed a set of six microarray experiments involving the use of a "regular" (unamplified) microarray experimental protocol and two different RNA amplification protocols. Based on their ability to identify differentially expressed genes and assuming that the results from the regular protocol are correct, our analyses demonstrated that both amplification protocols achieved reproducible and reliable results. From the same amount of starting material, our results also indicated that more amplified RNA can be obtained using conventional second-strand cDNA synthesis than from the combination of SMART and PCR. When the critical issue is the amount of starting RNA, we recommend the conventional second-strand cDNA synthesis as the preferred amplification method.

Pitfalls of Quantitative Real-Time Reverse-Transcription Polymerase Chain Reaction
Stephen A Bustin and Tania Nolan
Centre for Academic Surgery Institute of Cell and Molecular Science Barts and
The London Queen Mary’s School of Medicine and Dentistry, University of London; London, UK;

Stratagene Europe, Amsterdam, Netherlands

Polymerase chain reaction (PCR)-based assays can target either DNA (the genome) or RNA (the transcriptome).Targeting the genome generates robust data that are informative and, most importantly, generally applicable. This is because the information contained within the genome is contextindependent; i.e., generally, every normal cell contains the same DNA sequence the same mutations and polymorphisms. The transcriptome, on the other hand, is contextdependent; i.e., the mRNA complement and level varies with physiology, pathology, or development.This makes the information contained within the transcriptome intrinsically flexible and variable. If this variability is combined with the technical limitations inherent in any reverse-transcription (RT)-PCR assay, it can be difficult to achieve not just a technically accurate but a biologically relevant result. Template quality, operator variability, the RT step itself, and subjectivity in data analysis and reporting are just a few technical aspects that make real-time RT-PCR appear to be a fragile assay that makes accurate data interpretation difficult.There can be little doubt that in the future, transcriptome-based analysis will become a routine technique. However, for the time being it remains a research tool, and it is important to recognize the considerable pitfalls associated with transcriptome analysis, with the successful application of RT-PCR depending on careful experimental design, application, and validation.

An excellent overviews on quantitative kinetic RT-PCR !!!

Absolute quantification of mRNA using  real-time reverse transcription PCR assays
Bustin SA  
Journal of Molecular Endocrinology 25: 169-193 ( 2000)


The reverse transcription polymerase chain reaction (RT-PCR) is the most sensitive method for the detection of low-abundance mRNA, often obtained from limited tissue samples. However, it is a complex technique, there are substantial problems associated with its true sensitivity, reproducibility and specificity and, as a quantitative method, it suffers from the problems inherent in PCR. The recentintroduction of fluorescence-based kinetic RT-PCR procedures significantly simplifies the process of producing reproducible quantification of mRNAs and promises to overcome these limitations. Nevertheless, their successful application depends on a clear understanding of the practical problems, and careful experimental design, application and validation remain essential for accurate quantitative measurements of transcription. This review discusses the technical aspects involved, contrasts conventional and kinetic RT-PCR methods for quantitating gene expression and compares the different kinetic RT-PCR systems. It illustrates the usefulness of these assays by demonstrating the significantly different levels of transcription between individuals of the housekeeping gene family,glyceraldehyde-3-phosphate-dehydrogenase (GAPDH).


Quantification of mRNA using real-time reverse transcription PCR: trends and problems
by  Bustin SA.   J Mol Endocrinol. 2002 29: 23-29   Review.

The fluorescence-based real-time reverse transcription PCR (RT-PCR) is widely used for the quantification of steady-state mRNA levels and is a critical tool for basic research, molecular medicine and biotechnology. Assays are easy to perform, capable of high throughput, and can combine high sensitivity with reliable specificity. The technology is evolving rapidly with the introduction of new enzymes, chemistries and instrumentation. However, while real-time RT-PCR addresses many of the difficulties inherent in conventional RT-PCR, it has become increasingly clear that it engenders new problems that require urgent attention. Therefore, in addition to providing a snapshot of the state-of-the-art in real-time RT-PCR, this review has an additional aim: it will describe and discuss critically some of the problems associated with interpreting results that are numerical and lend themselves to statistical analysis, yet whose accuracy is significantly affected by reagent and operator variability.

One-Step RT-PCR without Initial RNA Isolation Step for Laser-Microdissected Tissue Sample
Kiyoshi KOBAYASHI1)2), Hiroyuki UTSUMI2), Miyoko OKADA2), Tetsuya SAKAIRI2),
Itsuko IKEDA2), Manami KUSAKABE2) and Shirou TAKAGI2)
1) Discovery Technology Laboratory, Mitsubishi Pharma Co.
2) Toxicology Laboratory, Mitsubishi Pharma Co.

Journal of Veterinary Medical Science Vol. 65 (2003) , No. 8 August pp.917-919

One-step RT-PCR procedure without initial RNA extraction step is tested for laser microdissected tissue sample. Unfixed cryosections of liver and kidney tissue of male SD rats were cut using laser microdissection system and directly used as templates for RT-PCR study. To check the sensitivity, 5, 25, 125, and 625 hepatocytes were cut and put in PCR-tube. After DNase treatment and cDNA synthesis with pd(N)6 random primer, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNAs were amplified by 60 thermal cycles. GAPDH-specific bands were observed at as few as 25 hepatocytes. Specificity of this procedure was tested for hepatocytes, renal tubular epithelium and glomerular tissue using albumin PCR primers. Approximately 250 cells were cut and albumin cDNA was amplified as described above. Albumin specific band was observed only in hepatocytes sample. To apply this approach to quantitative PCR, various numbers of hepatocytes were cut and put in 0.2 mL PCR tube. After reverse transcription and 10 cycles of GAPDH cDNA amplification by regular thermal-cycler, PCR solution was transferred to 96-well plate designed for real-time PCR system, and further 40 cycles were performed. As a result, GAPDH cDNAs were successfully amplified with a good correlation between the number of template hepatocytes and the intensity of PCR signal. From these results, we concluded this approach would be very useful for the expression analysis of microdissected pathology samples.

Optimized protocol for linear RNA amplification and application to gene
expression profiling of human renal biopsies.
Scherer A, Krause A, Walker JR, Sutton SE, Seron D, Raulf F, Cooke MP.
Novartis Pharma AG, Basel, Switzerland.
Biotechniques. 2003 Mar;34(3):546-50, 552-4, 556.
Gene expression analysis using high-density cDNA or oligonucleotide arrays is a rapidly emerging tool for transcriptomics, the analysis of the transcriptional state of a cell or organ. One of the limitations of current methodologies is the requirement of a relatively large amount of total or polyadenylated RNA as starting material. Standard array hybridization protocols require 5-15 micrograms labeled RNA. To obtain these quantities from small amounts of starting RNA material, RNA can be amplified in a linear fashion. Here we introduce an optimized protocol for rapid and easy-to-use amplification of as little as 1 ng total RNA. Our analysis shows that this method is linear and highly reproducible and that it preserves similarities as well as dissimilarities between normal and disease-related samples. We applied this technique to the RNA expression profiling of human renal allograft biopsies with normal histology and compared them to the profiles of renal biopsies with histological evidence of chronic transplant nephropathy or chronic rejection. Among others, complement component C1r was found to be significantly up-regulated in chronic rejection and chronic transplant nephropathy biopsies compared to normal samples, while fructose-1,6-biphosphatase showed lower-than-normal expression.