real-time PCR optimisation (2)
More qPCR optimisation papers: Manuals for optimising your qPCR: Troubleshooting and Talks: qPCR Satellite
Symposium
Optimisation, Normalisation & Standardisation 10-11th March 2005 http://leipzig05.gene-quantification.info
Richie Soong and Andra´s Ladanyi Department of Pharmacology and Toxicology, University of Alabama at Birmingham, Birmingham, AL 35294; Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA 02115 Clinical Chemistry. 2003;49:973-976 Because
of their low detection limits, PCR and the adapted kinetic (real-time
quantitative) PCR have been used extensively for the detection and
quantification of nucleic acids (1)(2)(3). However, at the detection
limits of an assay, mathematical models (4)(5)(6) and experimental
evidence (7)(8)(9)(10)(11)(12) have shown that nucleic acids are
detected inconsistently and quantified imprecisely. With this potential
unreliability, it is essential that indicators be used to identify the
concentration at which analysis is occurring to ensure the accuracy of
results. Conventional PCR indicators, however, currently provide only
an indirect assessment of the concentration at which analysis occurs.
The total nucleic acid content of a sample, commonly used to define the
adequacy of sample loading, does not precisely reflect specific gene
concentrations. Reference (control or housekeeping) genes, qualitative
detection of which is often used to validate assay and sample
integrity, are often present at concentrations different from those of
the target genes of interest (13). In kinetic PCR, the inclusion of
fluorescent probes allows the monitoring of reaction kinetics, which
enables the measurement of a crossing point (CP), or cycle threshold,
at a partial cycle number at which fluorescence becomes detectable
above background signal (14). CP values are linearly proportional to
gene copy number in an inverse logarithmic relationship (14)(15), and
this correlation is central to algorithms for determining gene quantity
(16)(17). In this study, we hypothesized that CP values might be more
accurate indicators of the reliability of an analysis than conventional
indicators because of their direct relationship with gene
concentration. The results of this study provide experimental evidence
to support this hypothesis; we also describe models for use of CP
values as indicators for assessing the reliability of analysis.
To test our hypothesis, we simulated the analysis of low concentrations of the putative colorectal micrometastasis marker cytokeratin 20 (CK20) (16) and the reference genes porphobilinogen deaminase (PBGD) and ß2-microglobulin (ß2M) and compared the capabilities of conventional indicators (total nucleic acid content and qualitative reference gene detection) and CP values to identify samples with unreliable analysis. Quantitative assessment of PML-RARa and BCR-ABL by two real-time PCR instruments: multiinstitutional laboratory trial. Bolufer P, Colomer D, Gomez MT, Martinez J, Gonzalez SM, Gonzalez M, Nomdedeu J, Bellosillo B, Barragan E, Lo-Coco F, Diverio D, Hermosin L, Garcia-Marco J, De Juan MD, Barros F, Romero R, Sanz MA. Molecular Biology, Department of Medical Biopathology, Hospital Universitario La Fe, Avda Campanar 21, 46009 Valencia, Spain. Clin Chem. 2004 Jun;50(6): 1088-1092 The
recent introduction on the market of instruments for real-time PCR has
prompted the development of quantitative assays for the most common
fusion transcripts detectable in hematologic malignancies. However,
because the ABI PRISM apparatus (ABI; Applied Biosystems) was the first
available instrument for real-time PCR, most of the methods developed
for the ABI PRISM use TaqMan probe chemistry (1)(2)(3). With the
introduction of other real-time PCR instruments, such as the
LightCycler (LC; Roche), other methods have been described
(4)(5)(6)(7). The instruments differ in several respects, including the
light sources and the approach to acquisition of fluorescence data. Few
reports have compared the results obtained with different types of
real-time PCR instruments (8). To the best of our knowledge, no such
multicenter studies with common calibrators and common methods have
been reported.
In the present study we analyzed the results obtained with two of the more widely used instruments for real time PCR, i.e., the ABI and LC, for amplifying two rearrangements frequently detectable in human leukemia, the BCR-ABL and PML-RARa fusion genes. For BCR-ABL several quantitative methods have been established for both instruments (3)(4)(5)(6)(7), whereas for PML-RARa most of the quantitative methods have been developed for the ABI PRISM. |
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A
sensitive and quantitative single-tube real-time reverse
transcriptase-PCR for detection
of enteroviral RNA.
Mohamed N, Elfaitouri A, Fohlman J, Friman G, Blomberg J. Section of Virology, Department of Medical Sciences, Uppsala University, Uppsala 75185, Sweden. J Clin Virol. 2004 Jun;30(2): 150-156. BACKGROUND:
Enteroviruses (EVs) are significant human pathogens. Rapid and
sensitive diagnostic
techniques are desirable. OBJECTIVES:
To develop a quantitative
single-tube real-time reverse transcription-polymerase chain reaction
(RT-PCR) for
human enterovirus ribonucleic acid (RNA) (QPCR), with protection
against
amplimer contamination. STUDY DESIGN: The method was evaluated with
serial
dilutions of EV, 62 cerebrospinal fluid (CSF) specimens from meningitis
patients, and the third and fourth European Union Concerted Action
Enterovirus
Proficiency Panels. A commercial EV PCR test was run in parallel.
RESULTS: Optimisation included RNA extraction procedure, design and concentrations of primers and probes from the 5' non-coding region as well as recombinant Thermus thermophilus polymerase (rTth), Mn(OAc)(2) and thermolabile UNG concentrations. Of 62 CSF samples from cases of meningitis submitted for QPCR testing, 34 (76%) and 21 (47%) were positive by QPCR and a commercial EV RNA detection kit, respectively. The detection limit of QPCR was 0.001 TCID(50)/ml (50% tissue culture-infective dose per millilitre) for a coxsackievirus B2 preparation and <10 copies of a plasmid containing coxsackievirus B2 complementary deoxyribonucleic acid (cDNA). The relation between threshold cycle (C(t)) and amount of virus was linear (r = 0.99) over a range of 10(-3) to 10(4) TCID(50)/ml of coxsackievirus B2. CONCLUSIONS: The QPCR method allows a large number of samples to be screened rapidly. Its sensitivity, simplicity, and reproducibility make it a suitable tool for the routine laboratory. Enhanced
analytical sensitivity of a quantitative PCR for CMV using a modified
nucleic-acid
extraction procedure.
Ferreira-Gonzalez A, Yanovich S, Langley MR, Weymouth LA, Wilkinson DS, Garrett CT. Department of Pathology, Medical College of Virginia Campus of Virginia Commonwealth University, Richmond, Virginia 23298-0248, USA. J Clin Lab Anal. 2000;14(1): 32-37. Accurate
and
rapid diagnosis of CMV disease in immunocompromised individuals remains
a
challenge. Quantitative polymerase chain reaction (QPCR) methods for
detection
of CMV in peripheral blood mononuclear cells (PBMC) have improved the
positive and
negative predictive value of PCR for diagnosis of CMV disease. However,
detection of CMV in plasma has demonstrated a lower negative predictive
value
for
plasma as compared with PBMC. To enhance the sensitivity of the QPCR
assay for
plasma specimens, plasma samples were centrifuged before nucleic-acid
extraction and
the extracted DNA resolubilized in reduced volume. Optimization of the
nucleic-acid extraction focused on decreasing or eliminating the
presence of inhibitors in
the pelleted plasma. Quantitation was achieved by co-amplifying an
internal
quantitative standard (IS) with the same primer sequences as CMV. PCR
products
were detected by hybridization in a 96-well microtiter plate coated
with a CMV or IS
specific probe. The precision of the QPCR assay for samples prepared
from
untreated and from pelleted plasma was then assessed. The coefficient
of
variation for both types of samples was almost identical and the
magnitude
of the coefficient of variations was reduced by a factor of ten if the
data were
log transformed. Linearity of the QPCR assay extended over a 3.3-log
range for
both types of samples but the range of linearity for pelleted plasma
was 20 to
40,000 viral copies/ml (vc/ml) in contrast to 300 to 400,000 vc/ml for
plasma. Thus,
centrifugation of plasma before nucleic-acid extraction and
resuspension of
extracted CMV DNA in reduced volume enhanced the analytical sensitivity
approximately tenfold over the dynamic range of the assay.
Quantitation of
residual WBCs
in
filtered blood components by high-throughput, real-time
kinetic
PCR.
Lee TH, Wen L, Chrebtow V, Higuchi R, Watson RM, Sninsky JJ, Busch MP. Molecular Biology Blood Centers of the Pacific, Irwin Center, Research Division, San Francisco, California 94118, USA. Transfusion. 2002 Jan;42(1): 87-93. BACKGROUND:
The effort to eliminate transfusion complications
associated with WBCs has led to the widespread use
of filters able to reduce WBC
concentrations to <or =0.1 WBC per microL
blood. This has necessitated sensitive QC
methods to quantitate residual WBCs in
filtered units. One fast, effective method
is DNA amplification
using real-time kinetic PCR (kPCR).
STUDY DESIGN AND METHODS: Two methods of preparation of standards were compared and used for the optimization of quantitative kPCR. The first involved spiking genomic DNA cell lysate into a diluent, followed by a series of 1 in 10 dilutions. The second involved spiking serial 1 in 10 dilutions of WBCs into twice-filtered fresh whole blood. Two hundred fifty filtered frozen whole-blood samples were amplified in duplicate to show the kPCR assay's reproducibility. Another 359 filtered frozen whole blood samples were used to compare data from kPCR with data from a standard PCR protocol using (32)P-labeled probe and autoradiography. All specimens were amplified for conserved HLA DQ(alpha) sequences. RESULTS: Standards prepared by both methods gave reproducible and equivalent results. Quantitation of standards representing a dynamic range of 8 x 10(o) to 8 x 10(5) WBCs per mL, yielded standard deviations ranging from 0.59 cycle to 1.04 cycles (a one-cycle increase is equivalent to a twofold increase in WBC concentration). The scatter graph of the 250 samples tested in duplicate by kPCR generated a slope of 1.0122 and an R(2) value of 0.9265. The comparison of kPCR and (32)P-probe hybridization results on 359 clinical samples gave a scatter-graph slope of 0.9428 and an R(2) value of 0.8718, indicating excellent agreement of the methods over a 4-log dynamic range. CONCLUSION: kPCR is a high-throughput, sensitive assay that could prove useful in routine quality assurance of the WBC reduction process. Influence of
reagents formulation on real-time PCR parameters.
Real-time polymerase chain
reaction (PCR) techniques are increasingly used to quantify target
sequences for
diagnostic and research purposes. Due to its 'quantitative' character,
it is
very important to determine the variability of this technique
correlating with
several experimental conditions. The objective of this study was to
analyse the
effect of manufacturing lots of PCR reagents on two main PCR
parameters,
specificity and sensitivity. For this study, we used four different
amplicons, using
either mouse genomic DNA or viral DNA. Although a PCR product could be
obtained in
any of the conditions, we observed that there are relevant variations
in
sensitivity depending on the reagents formulation. We conclude that
different lots of
reagents may determine the analytical performance of PCR assays
indicating that reagents testing are of special importance when the PCR
protocol
is used for quantitative purposes.Burgos JS, Ramirez C, Tenorio R, Sastre I, Bullido MJ. Centro de Biologia Molecular Severo Ochoa - CSIC Universidad Autonoma de Madrid, Cantoblanco, Madrid, Spain. Mol Cell Probes. 2002 Aug;16(4): 257-260 High-sensitivity
quantitative PCR platform.
Real-time PCR methods have become
widely used within the past few years. However, real-time PCR is rarely
used to study chronic diseases with low pathogen loads, presumably
because
of insufficient sensitivity. In this report, we developed an integrated
nucleic
acid isolation and real-time PCR platform that vastly improved the
sensitivity of the quantitative detection of the intracellular
bacterium, Chlamydia spp., by fluorescence resonance energy transfer
real-time PCR.
Determinants of the overall detection sensitivity were analyzed by
extracting nucleic
acids from bovine milk specimens spiked with low amounts of chlamydial
organisms.
Nucleic acids were optimally preserved and recovered by collection in
guanidinium stabilization buffer, binding to a matrix of glass fiber
fleece, and elution
in low volume. Step-down thermal cycling and an excess of hot-start Taq
polymerase vastly improved the robustness and sensitivity of the
real-time PCR
while essentially maintaining 100% specificity. The amplification of
Chlamydia 23S
rRNA allowed for the differentiation of chlamydial species and was more
robust at low target numbers than amplification of the omp1 gene. The
best
combined method detected single targets per a 100-microL specimen
equivalent in
a 5-microL real-time PCR input. In an initial application, this
high-sensitivity
real-time PCR platform demonstrated a high prevalence of chlamydial
infection
in cattle.DeGraves FJ, Gao D, Kaltenboeck B. Auburn University, Auburn, AL, USA. Biotechniques. 2003 Jan;34(1): 106-115 Real-time
quantitative RT-PCR after laser-assisted cell picking.
The present study describes a
technique for quantitation of mRNA in a few isotypic cells obtained
from an
intact organ structure by combining laser-assisted cell picking and
real-time PCR. The microscopically controlled lasering of selected
cells in
stained tissue sections was applied to lung alveolar macrophages, which
are
unique in that they can alternatively be gathered as a pure cell
population
from intact lungs by bronchoalveolar lavage as a reference technique.
TNF-alpha was chosen as the transcriptionally inducible target gene to
be
quantified in alveolar macrophages of control rat lung, as well as low-
and
high-challenge lungs stimulated by endotoxin and IFN-gamma
nebulization. Online
fluorescence detection for quantitation of theFink L, Seeger W, Ermert L, Hanze J, Stahl U, Grimminger F, Kummer W, Bohle RM. Department of Pathology, Justus-Liebig-Universitat Giessen, Germany. Nat Med. 1998 Nov;4(11): 1329-1333 number of amplified copies was based on 5' nuclease activity of Taq polymerase cleaving a sequence-specific dual-labeled fluorogenic hybridization probe. A pseudogene-free sequence of PBGD served as an internal calibrator for comparative quantitation of target. A quick procedure and minimized loss of template were achieved by avoiding RNA extraction, DNase digestion and nested-PCR. Using this approach, we demonstrated dose-dependent manifold upregulation of the ratio of TNF-alpha mRNA copies per one copy of PBGD mRNA in alveolar macrophages of the challenged lungs. The quantitative data obtained from laser-picked alveolar macrophages were well matched with those of lavaged alveolar macrophages carried out in parallel. We suggest that this new combination of laser-assisted cell picking and real-time PCR has great promise for quantifying mRNA expression in a few single cells or oligocellular clusters in intact organs, allowing assessment of transcriptional regulation in defined cell populations.
We have developed a new package of
computer programs and algorithms for different PCR applications,
including allele-specific PCR, multiplex PCR, and long PCR. The package
is included
in the upcoming VectorNTI suite software and attempts to incorporate
most of
the current knowledge about PCR primer design. A wide range of primer
characteristics is available for user manipulation to provide improved
efficiency and
increased flexibility of primer design. Toaccelerate the primer
calculations, we have optimized algorithms using recent advances in
computer science such
as dynamic trees and lazy evaluation. Proper structural organization of
input
parameters provides further program acceleration. New Vector NTI
primer design software allows calculations of primer pairs for long PCR
amplification of 120-kb genomic DNA in 5 min under most stringent input
parameters
and clustering 435 primer pairs for multiplex PCR within 30 min on a
standard
Pentium III PC. Our program allows the user to take advantage of
molecule
annotation by applying different kinds of filtering features during PCR
primer design.
Optimisation of PCR
reactions using primer chessboarding.
In-house polymerase chain reaction
(PCR) assays are now an integral part of the work of most diagnostic
microbiological laboratories. Despite the availability of commercial
reagent
'master-mixes' of some PCR reagents, the optimisation of primers still
poses a significant
problem. Here, we describe a simple method to assess the concentration
of primer
needed in single round, multiplex, nested and 'real-time' PCR
procedures.Gunson R, Gillespie G, F Carman W. West Of Scotland Specialist Virology Centre, A Member of the UK Clinical Virology Network, Gartnavel General Hospital, 1053 Great Western Road, G12 OYN, Glasgow, UK. J Clin Virol. 2003 26(3): 369-373. Teruo Ikedaa, Masaru Murakamib, Masayuki Funaba Veterinary Immunology and Immunopathology 100 (2004) 1–5 Three
methods to quantify gene
transcript levels in mast cells, real-time RT-PCR, competitive RT-PCR
and
conventional RT-PCR analyses, were compared. Linear regression analysis
on five
gene transcripts revealed that the mRNA levels measured by real-time
RT-PCR analysis were minimally correlated with those by conventional
RT-PCR
analysis. In addition, differences in the mRNA level between samples
measured by
conventional RT-PCR analysis were smaller than those by real-time
RT-PCR
analysis, suggesting that conventional RT-PCR analysis is less
sensitive at measuring
mRNA levels. Results from competitive RT-PCR analysis correlated
closely with
those from real-time RT-PCR analysis. When the differences in mRNA
level between
samples are relatively smaller, however, the correlation tended to be
weaker.
Real-time RT-PCR analysis has higher reliability, but is expensive. In
contrast, competitive RT-PCR analysis is inexpensive, but is weaker at
detecting smaller differences in gene transcript level between samples.
Therefore,
the most appropriate analytical method to measure mRNA levels should be
chosen, depending on the experimental conditions.
Detection of RNA viruses by
reverse transcription (RT)-PCR has proven to be a useful approach for
the diagnosis
of infections caused by many viral pathogens. However, adequate
controls are
required for each step of the RT-PCR protocol to ensure the accuracies
of
diagnostic test results. Heterologous competitor RNA can be used as a
control for a
number of different aspects of diagnostic RT-PCR. Competitor RNA can be
applied to
assessments of the efficiency of RNA recovery during extraction
procedures,
detection of endogenous RT-PCR inhibitors that could lead to
false-negative
results, and quantification of viral template in samples used for
diagnosis;
competitor RNA can also be used as a positive control for the RT-PCR.
In the
present study, heterologous competitor RNA was synthesized by a method
that uses
two long oligonucleotide primers containing primer binding sites for
RT-PCR
amplification of porcine reproductive and respiratory syndrome virus or
West
Nile virus. Amplification of the competitor RNA by RT-PCR resulted in a
product that was easily distinguished from the amplification product of
viral RNA
by agarose gel electrophoresis. Assessment of a variety of RNA samples
prepared
from routine submissions to a veterinary diagnostic laboratory found
that
either partial or complete inhibition of the RT-PCR could be
demonstrated for
approximately 20% of the samples. When inhibition was detected, either
dilution of the sample or RNA extraction by an alternative protocol
proved
successful in eliminating the source of inhibition.
Influence of DNA
polymerases on quantitative PCR results using TaqMan probe format in the
LightCycler instrument.
Kreuzer KA, Bohn A, Lass U, Peters UR, Schmidt CA. Abteilung fur Innere Medizin und Poliklinik m.S. Hamatologie und Onkologie, Medizinische Fakultat Charite der Humboldt-Universitat zu Berlin, Germany. Mol Cell Probes. 2000 Apr;14(2): 57-60. Real-time fluorescence polymerase chain reaction (PCR) techniques are increasingly used to quantitate target sequences for diagnostic and research purposes. Currently, the so called TaqMan probe chemistry is mostly used as fluorogenic system. This probe format is strictly dependent on the 5'-exonuclease activity of DNA polymerase as fragmentation of the probe during the reaction is essential for this assay. Based on our experience that dramatic differences in quantitative PCR results may be due to different DNA polymerases we performed a detailed comparison of 15 enzymes. We found that clear differences exist between polymerases of different manufacturers. Thus, three out of seven polymerases which were declared to possess 5'-exonuclease activity appeared to be completely unsuitable for this method while the remaining had significantly different reaction efficiencies. We conclude that different DNA polymerases may determine the entire analytical performance of TaqMan assays suggesting that DNA polymerase testing is of special importance when this probe format is used. Differential priming
of RNA templates during cDNA synthesis markedly affects
Quantitative competitive
reverse-transcriptase PCR is the most sensitive method for studying
gene expression. To
investigate whether the accuracy of the calculated target mRNA copy
number
is affected by the cDNA priming process, we utilized primers of
different
lengths, concentrations and primer sequences to prime cDNA synthesis
reactions.
Our results show a approximately 19-fold increase in the calculated
mRNA
copy number from cDNA synthesis reactions primed with random hexamers
(P<0.001,
n=4), and a approximately 4-fold increase in copy number with a
specific hexamer
(P<0.001, n=4) compared with that obtained with a
22-mer-sequence-specific primer.
The increase in calculated mRNA copy number obtained by priming cDNA
synthesis
with the shorter specific and non-specific primers could be explained
largely
by the synthesis of truncated standard cDNA molecules lacking a
requisite
binding site for amplification with PCR primers. Since these truncated
standard
cDNA molecules could not be amplified and standard RNA is used to
quantify
target mRNA copy number, this phenomenon resulted in overestimation of
target mRNA copy number. In conclusion, accurate determination of
target mRNA copy
number is most likely if a long specific antisense primer is used to
prime
cDNA synthesis.both accuracy and reproducibility of quantitative competitive reverse-transcriptase PCR. Zhang J, Byrne CD. Department of Clinical Biochemistry, University of Cambridge, Box 232, Level 4, Addenbrookes Hospital, Hills Road, Cambridge CB2 2QR, U.K. Biochem J. 1999 Jan 15;337 ( Pt 2): 231-41. Evaluation of
sense-strand mRNA amplification by comparative quantitative PCR.
INTRODUCTION: RNA amplification is
required for incorporating laser-capture microdissection techniques
into microarray assays. However, standard oligonucleotide microarrays
contain sense-strand 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 real-time PCR
(qPCR),
mimicking a two-color microarray assay.Goff LA, Bowers J, Schwalm J, Howerton K, Getts RC, Hart RP. W.M. Keck Center for Collaborative Neuroscience, Rutgers University, Piscataway, NJ 08854, USA. BMC Genomics. 2004 Oct 06;5(1):76. 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.
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.
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