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Critical Factors for Successful Real-Time PCR

High PCR efficiency enable more accurate quantification in real-time PCR

The first universal kit for quantitative, real-time, multiplex PCR

  Improved Indicators for Assessing the Reliability of Detection and Quantification by Kinetic PCR.
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.

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.
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
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.

High-sensitivity quantitative PCR platform.
DeGraves FJ, Gao D, Kaltenboeck B.
Auburn University, Auburn, AL, USA.
Biotechniques. 2003 Jan;34(1): 106-115
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.

Real-time quantitative RT-PCR after laser-assisted cell picking.
Fink 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
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 the
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.

Set of novel tools for PCR primer design.
Gorelenkov V, Antipov A, Lejnine S, Daraselia N, Yuryev A.
InforMax, Bethesda, MD, USA.
Biotechniques. 2001 Dec;31(6): 1326-1330.
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.
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.
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.

Reliability of RT-PCR methods for measuring relative gene expression in mast cells
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.

Reliability of RT-PCR methods for measuring relative gene expression in mast cells.
Ikeda T, Murakami M, Funaba M.
Research Institute of Biosciences, Azabu University, 1-17-71 Fuchinobe,
Sagamihara 229-8501, Japan.
Vet Immunol Immunopathol. 2004 Jul;100(1-2): 1-5.
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
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.
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.

Evaluation of sense-strand mRNA amplification by comparative quantitative PCR.
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.
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.
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.

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.