 mRNA Transcript Analysis: Reverse Transcription (3) mRNA Transcript Analysis: Reverse Transcription (1) mRNA Transcript Analysis: Reverse Transcription (2) RNA integrity
MOLECULAR
BIOLOGY: RNA lost in translation.
David Tollervey Nature Vol 440 23 March 2006 In any manufacturing process, quality control is crucial, and gene expression is no exception. A new pathway monitors mRNAs - the intermediaries of gene expression - and destroys faulty molecules. Reverse
transcription
using random pentadecamer primers increases yield and quality of
resulting cDNA:
Michael Stangegaard,
Inge Høgh Dufva, and Martin Dufva
Reverse transcription of
RNA is an invaluable method
for gene expression analysis by real-time PCR or microarray methods.
Random primers of varying lengths were compared with respect to their
efficiency of priming reverse transcription reactions. The results
showed that 15-nucleotide-long random oligonucleotides (pentadecamers)
consistently yielded at least 2-fold as much cDNA as did random
hexamers using either poly(A) RNA or an amplified version of messenger
RNA (aRNA) as a template. The cDNA generated using pentadecamers did
not differ in size distribution or the amount of incorporated label
compared with cDNA generated with random hexamers. The increased
efficiency of priming using random pentadecamers resulted in reverse
transcription of >80% of the template aRNA, while random hexamers
induced reverse transcription of only 40% of the template aRNA. This
suggests a better coverage of the transcriptome when using random
pentadecamers over random hexamers. Using the same amount of aRNA as
starting material, random pentadecamer-primed reactions resulted in
11-fold more genes being detected in whole transcriptome DNA microarray
experiments than random hexamer-primed reactions. The results indicate
that random pentadecamers can replace random hexamers in reverse
transcription reactions on both poly(A) RNA and amplified RNA,
resulting in higher cDNA yields and quality.
K. Swinson, M. Koban
Laboratory of Physiology, Richard N. Dixon Science Research Building, Department of Biology, Morgan State University, 1700 E. Cold Spring Lane, Baltimore, Maryland 21251, USA The labile nature of
RNA demands careful procedures for its extraction, purification, and
storage. Generally, RNA is solubilized in aqueous buffers or organic
solvents, or precipitated with alcohol and then kept at 20 8C or
colder. A commercially available product for RNA storage is FORMAzol
(Molecular Research Center). We began using FORMAzol because the
Application Notes from the Product Description sheet claims that
reverse-transcription (RT) is not inhibited so long as FORMAzol does
not exceed 5% (v/v) in the reaction mix. This is ostensibly more
convenient than having to precipitate RNA, resolubilizing it in water
or buffer, and then proceeding with RTPCR. However, amplicon yields for
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were poor when using
RNA directly from FORMAzol, even though its final concentration was
typically much less than 5%. By contrast, satisfactory RT-PCR products
were obtained with RNA stored frozen in water or that had been
resolubilized from alcohol precipitates. When RT-PCR was then performed
on ethanol-precipitated and resolubilized RNA from FORMAzol, yields of
GAPDH amplicons were acceptable. Although a revision to the FORMAzol
Product Description sheet is now available at the manufacturer’s
website (http://www.mrcgene.com/formazol.htm), if users of the product
implicitly follow the directions found in the package insert sheet—not
being aware of the inhibitory effects of formamide (the denaturant in
FORMAzol) — unsatisfactory results may be obtained from RT-PCR
experiments. It is suggested that FORMAzol only be used for RNA storage
and that RNA be precipitated with alcohol, washed, and resolubilized
prior to use.<>
Michael J. Wacker and
Michael P. Godard
Applied Physiology
Laboratory, University of Kansas, Lawrence, Kansas
Real-time reverse
transcription polymerase chain reaction (RT-PCR) is a commonly used
technique to analyze gene expression. There has been little research
conducted to test if Super-Script III quantitative one-step (reverse
transcription carried out in the same tube as PCR) and two-step
(reverse transcription carried out in a separate reaction) RT-PCR
systems provide similar real-time results. In this study, real-time
reactions were set up using the housekeeping genes glyceraldehyde
phosphate dehydrogenase (GAPDH), β2-microglobulin (B2M), and RNA
polymerase 2 subunit A (PolR2A). Reaction efficiencies were determined
by generating standard curves using total RNA isolated from human
skeletal muscle and brain. Reaction efficiencies ranged from 97.7
± 0.9% to 99.4 ± 1.8% for one-step and 98.0 ± 0.2%
to 102.6 ± 1.3% for two-step RT-PCR (R2 values for all reactions
≥ 0.995). The sensitivities of one-step and two-step methods, as
measured by cycle threshold values, were similar for GAPDH and B2M.
However, for the lesser expressed PolR2A mRNA there was a 5 cycle lower
threshold for one-step. In summary, both Super-Script III one-step and
two-step methods yield reaction efficiencies close to 100% and produce
similar, accurate, linear standard curves. However, using the one-step
method with gene-specific priming may be more sensitive for
quantification of certain genes such as PolR2A.
Reduced
Plasma RNA
Integrity in Nasopharyngeal Carcinoma Patients
Blenda C.K. Wong, K.C.
Allen Chan, Anthony T.C. Chan, Sing-Fai Leung, LisaY.S. Chan, Katherine
C.K. Chow, and Y.M. Dennis Lo
Purpose: Recent
research has shown the feasibility of detecting cell-free RNA markers
in human subjects. As elevated RNase activity has previously been
described in the circulation of cancer patients, we hypothesized that
cancer patients may have reduced plasma RNA integrity. In this study,
we used nasopharyngeal carcinoma (NPC) as amodel systemto test this
hypothesis. Experimental Design: Plasma RNA integrity was determined
using the ratio of the concentrations of transcript sequences
corresponding to the 3V to those from the 5V end of a housekeeping
gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Transcript
concentrations were measured using real-time quantitative reverse
transcription-PCR assays targeting the 5V and 3V regions. We analyzed
the plasma RNA integrity in 49 untreated NPC patients and 53 healthy
controls.We also assessed the plasma samples from 19 NPC patients
before and after radiotherapy to further show the clinical potential of
this marker. Results: The 3V to 5V GAPDH ratio was significantly lower
in the plasma of untreated NPC patients when compared with healthy
controls (0.0252 versus 0.0485, P = 0.024). Statistical analysis showed
that plasma GAPDH ratio was correlated with tumor stage but not with
sex and age. Moreover, 14 of 19 NPC patients (74%) showed significant
increase in the plasma GAPDH ratio following radiotherapy (P = 0.003).
All of these patients were in clinical remission after treatment.
Conclusions: Our findings suggest that NPC is associated with
disturbances in the integrity of cell-free circulating RNA, raising the
possibility that measurement of plasma RNA integrity may serve as a
usefulmarker for the diagnosis andmonitoring of malignant diseases.
Direct
quantification of
gene expression in homogenates of formalin-fixed, paraffin-embedded
tissues.
Wen Yang, Botoul Maqsodi, Yunqing Ma, Son Bui, Kimberly L. Crawford, Gary K. McMaster, Frank Witney, and Yuling Luo Genospectra, Inc.,
Fremont, CA, USA
BioTechniques 40:481-486 (2006) Formalin-fixed,
paraffin-embedded (FFPE) tissues represent an important source of
archival materials for gene expression profiling. We report here the
development of a modified branch DNA assay that allows direct
quantification of messenger RNA (mRNA) transcripts in homogenates from
FFPE tissue sections without the need for RNA isolation and reverse
transcription into cDNA. Formalin fixation essentially has no effect on
the branch DNA assay, and RNA degradation only marginally reduces the
signal by 2- to 3-fold. Under the same conditions, formalin fixation
and RNA degradation greatly reduces real-time reverse transcription PCR
(RT-PCR) efficiency, reducing signals by as much as 15- and 1400-fold,
respectively. Although both technologies can generate biologically
meaningful expression profiles from FFPE human lung tumor specimens,
the branch DNA assay is more sensitive than real-time RT-PCR under the
conditions tested. Our results therefore suggest that the branch DNA
assay is an ideal tool for retrospective analysis of gene expression in
archival tissues.
Quantification
of mRNA
in Whole Blood by Assessing Recovery of RNA and Efficiency of cDNA
Synthesis.
Masato Mitsuhashi,
Shigeru Tomozawa, Katsuya Endo, and Atsushi Shinagawa
Background: Current gene
expression analysis relies on the assumption that the isolated RNA
represents all species of mRNA in proportions equal to those in the
original materials. No system is available for absolute quantification
of mRNA. Methods: We applied whole blood to 96-well
filterplates to trap leukocytes. Lysis buffer containing cocktails of
specific reverse primers and known concentrations of synthetic external
control RNA (RNA34) was added to filterplates, and cell lysates were
transferred to oligo(dT)-immobilized microplates for hybridization. We
then synthesized the cDNA in the oligo(dT)-immobilized microplates from
these primer sites and used the cDNA for real-time PCR. RNA34 acted as
a universal control, and gene amplification results were converted to
quantities of mRNA per microliter of whole blood after the recovery of
RNA34 in each sample was determined. Results: Under fully optimized
conditions, both added RNA34 and native mRNA species exhibited 10%
recovery from whole blood to real-time PCR. When whole blood was
stimulated ex vivo, changes in gene expression as low as 30%–40% were
detected with statistical significance, and the experimental CVs
were low (10%–20%). Conclusion: This new system to estimate mRNA copies
per microliter of whole blood may allow standardization of
gene-expression–based molecular diagnostics.
Application
of in
situ
ductal perfusion to facilitate isolation of high-quality RNA from mouse
pancreas.
Anne E. Mullin, Galina Soukatcheva, C. Bruce Verchere, and Janet K. Chantler University of British
Columbia, Vancouver and Child and Family Research Institute, Vancouver,
BC, Canada
A technique to isolate
high-quality intact RNA from murine pancreas is described. This
technique involves in situ ductal perfusion of the pancreas with an
RNase inhibitor prior to removal of the organ for RNA extraction. In
this way, the pancreatic RNases are inhibited in situ allowing good
yields of intact RNA, suitable for studies on pancreatic gene
transcription by real-time PCR or microarray anaysis, to be obtained in
a reliable way.
Noh Jin Park, Yang Li,
Tianwei Yu, Brigitta M.N. Brinkman, David T. Wong
Background: We have
previously shown that human mRNAs are present in saliva and can be used
as biomarkers of oral cancer. In this study, we analyzed the integrity,
sources, and stability of salivary RNA. Methods: We measured the
integrity of salivary RNA with reverse transcription followed by PCR
(RT-PCR) or RT-quantitative PCR (RT-qPCR). To study RNA entry sites
into the oral cavity, we used RT-PCR analysis of salivary RNA from the
3 major salivary glands, gingival crevice
fluid,
and desquamated oral epithelial cells. We measured stability of the
salivar bneta-actin RNA by RT-qPCR of salivary RNA incubated at room
temperature for different periods of time. We measured RNA association
with other macromolecules by filtering saliva through pores of
different sizes before performing RT-qPCR. To assess RNA–macromolecule
interaction, we incubated saliva with Triton X-100 for different
periods of time before performing RT-qPCR. Results: In most cases, we
detected partial- to fulllength salivary mRNAs and smaller amounts of
middle and 3 gene amplicons compared with the 5. RNA was present in
all oral fluids examined. Endogenous salivary beta-actin
mRNA degraded more slowly than exogenous -actin mRNA, with half-lives
of 12.2 and 0.4 min, respectively (P <0.001). Salivary RNA could not
pass through 0.22 or 0.45 m pores. Incubation of saliva with Triton
X-100 accelerated degradation of salivary RNA. Conclusions: Saliva
harbors both full-length and partially degraded forms of mRNA. RNA
enters the oral cavity from different sources, and association with
macromolecules may protect salivary RNA from degradation.
Del Aguila EM, Dutra MB,
Silva JT, Paschoalin VM.
Departamento de BioquÃmica, Instituto de
QuÃmica, Universidade Federal do Rio de
Janeiro, Centro de Tecnologia, Bloco A, Lab. 545, CEP 21949-900, Rio de
Janeiro
(RJ), Brazil.
BMC Mol. Biol. 2005 Apr 156(1):9. Preparation of RNA free from DNA is a critical step before performing RT-PCR assay. Total RNA isolated from several sources, including those obtained from Saccharomyces cerevisiae, using routine methodologies are frequently contaminated with DNA, which can give rise to amplification products that mimic the amplicons expected from the RNA target. We investigated the efficiency of two DNase I based protocols for eliminating DNA contaminations from RNA samples obtained from yeast cells. Both procedures are very efficient in eliminating DNA contamination from RNA samples and entail three main steps, which involve treating of RNA samples with DNase I, inhibition of the enzyme by EDTA and its subsequent inactivation at 65 degrees C. The DNase I treated samples were further purified with phenol: chloroform followed by precipitation with ice-cold ethanol (protocol I) or, alternatively, they were directly used in RT-PCR reactions (protocol II). Transcripts from ACT1, PDA1, CNA1, CNA2, TPS1 and TPS2 analyzed after each treatment showed that all mRNAs tested can be amplified if total RNA was extracted and purified after DNase I treatment, however, only TPS1, TPS2 and ACT1 mRNAs were amplified without extraction/purification step. Although more laborious and requiring a higher initial amount of material, the inclusion of an extraction and purification step allows to prepare RNA samples that are free from DNA and from low molecular contaminants and can be applied to amplify any Saccharomyces cerevisiae mRNA by RT-PCR. John Ashkenas, James W.
Dennis, and Chi Yip Ho
Samuel Lunenfeld
Research Institute, Toronto, ON, Canada
BioTechniques 39:69-73 (July 2005) Reverse transcription
PCR (RT-PCR) is prone to false
positives when contaminating DNA molecules are present at the start of
a reaction. Contaminants that derive from earlier work using a given
primer pair (carryover PCR products) are of particular concern when
those primers are used routinely, as in clinical diagnostics or
environmental monitoring. In addi-tion, contamination by genomic DNA
can significantly interfere with quantitative and quali-tative analysis
of RNAs by RT-PCR. Here we describe contaminant restriction (ConR), a
method that can be used to neutralize carryover and genomic DNA
contamination in RT-PCR studies. Restriction enzymes (REs) added to the
amplification cocktail cleave contaminant DNA molecules while sparing
the intended target nucleic acid. Restriction, reverse tran-scription,
and amplification steps all take place in the same sealed vessel, thus
avoiding any danger of recontamination. ConR eliminates carryover
contamination in PCR without compromising target sequence
amplification. Because the method is effective against both genomic and
carryover contamination, it can be employed routinely in one-step
RT-PCR, whatever the RNA target or the nature of the potential DNA
contaminant. A variation of this decontamination method, amplicon
primer site restriction (APSR), is effective specifically against
carryover contamination. APSR, unlike ConR, can be applied during
PCR-based amplification of DNA target molecules.
Critical
points of DNA
quantification by real-time PCR – effects of DNA extraction method and
sample matrix on quantification of genetically modified organisms.
Katarina Cankar, Dejan
Štebih, Tanja Dreo, Jana Žel and Kristina Gruden
Background: Real-time
PCR is the technique of choice for nucleic acid quantification. In the
field of detection of genetically modified organisms (GMOs)
quantification of
biotech products may be required to fulfil legislativerequirements.
However, successful quantification depends crucially on
the quality of the sample DNA analyzed. Methodsfor GMO detection are
generally validated on certified reference
materials that are in the form of powdered grainmaterial, while
detection in routine laboratories must be performed on
a wide variety of sample matrixes. Due to foodprocessing, the DNA in
sample matrixes can be present in low amounts
and also degraded. In addition, molecules of plantorigin or from other
sources that affect PCR amplification of samples
will influence the reliability of the quantification.Further, the wide
variety of sample matrixes presents a challenge for
detection laboratories. The extraction method mustensure high yield and
quality of the DNA obtained and must be carefully
selected, since even components of DNAextraction solutions can
influence PCR reactions. GMO quantification is
based on a standard curve, therefore similarity of PCR efficiency for
the sample and standard reference material is a
prerequisite for exact quantification. Little information on the
performance of real-time PCR on samples of different
matrixes is available. Results: Five commonly used DNA extraction
techniques were compared and
their suitability for quantitative analysis was assessed. The effect of
sample matrix on nucleic acid
quantification was assessed by comparing 4 maize and 4 soybeanmatrixes.
In addition 205 maize and soybean samples from routine
analysis were analyzed for PCR efficiency to assessvariability of PCR
performance within each sample matrix. Together with
the amount of DNA needed for reliablequantification, PCR efficiency is
the crucial parameter determining the
reliability of quantitative results, therefore it waschosen as the
primary criterion by which to evaluate the quality and
performance on different matrixes and extractiontechniques. The effect
of PCR efficiency on the resulting GMO content
is demonstrated.Conclusion: The crucial influence of extraction
technique and sample
matrix properties on the results of GMOquantification is demonstrated.
Appropriate extraction techniques for
each matrix need to be determined to achieveaccurate DNA
quantification. Nevertheless, as it is shown that in the
area of food and feed testing matrix with certainspecificities is
impossible to define strict quality controls need to
be introduced to monitor PCR. The results of our study are
also applicable to other fields of quantitative testing by
real-time PCR.
Leslie Cler, Dawei Bu, Cheryl Lewis, David Euhus Hamon Center for Therapeutic Oncology Research, UT Southwestern Medical Center at Dallas, 5323 Harry Hines Boulevard, Dallas, TX 75390-9155, USA
Translational protocols
in cancer and carcinogenesis
often require isolation of genomic DNA from paucicellular clinical
samples. DNA extraction methods for PCR-based applications should
optimize the recovery of amplifiable DNA. We compared five methods for
DNA extraction in paucicellular epithelial and lymphocyte samples using
proportion of extractions producing amplifiable DNA and mean real-time
PCR Ct values for GAPDH as the endpoint measures. The methods included
solid-phase DNA adsorption (QIAamp), sequential protein and DNA
precipitation (Puregene), magnetic bead adsorption (Dynabeads),
phenol–chloroform extraction, and single-step proteinase K digestion.
In general, the performance of the three commercial kits was superior
to either phenol–chloroform extraction or single-step proteinase K
digestion. However, QIAamp and Puregene produced amplifiable DNA more
frequently than Dynabeads for starting cell numbers !50,000. GAPDH Ct
values for QIAamp extractions showed the greatest dynamic range and the
best linearity across the range of starting cell numbers, but QIAamp
was not statistically significantly superior to Puregene. Of the three
commercial kits, Puregene is the least expensive. QIAamp and Puregene
DNA extraction methods are well-suited for the preparation of
paucicellular clinical samples for PCR-based assays.
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