 mRNA Transcript Analysis: Reverse Transcription (2) mRNA Transcript Analysis: Reverse Transcription (1) mRNA Transcript Analysis: Reverse Transcription (3) RNA integrity
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. 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. 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 JOURNAL OF BIOMOLECULAR TECHNIQUES, VOLUME 15, ISSUE 3, SEPTEMBER 2004 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 Summary 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
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 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
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.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. |
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