New papers for optimising and standardising of qPCR / RT-qPCR:
The reproducibility of biomedical research: Sleepers awake!
Stephen A. Bustin,
Biomolecular Detection and Quantification, Volume 2, December 2014, Pages 35–42
There is increasing concern about the reliability of biomedical research, with recent articles suggesting that up to 85% of research funding is wasted. This article argues that an important reason for this is the inappropriate use of molecular techniques, particularly in the field of RNA biomarkers, coupled with a tendency to exaggerate the importance of research findings.
Critical Review -- Real-time PCR detection chemistry
Navarro E, Serrano-Heras G, Castaño MJ, Solera J
Clin Chim Acta. 2015 439: 231-250
Real-time PCR is the method of choice in many laboratories for diagnostic and food applications. This technology merges the polymerase chain reaction chemistry with the use of fluorescent reporter molecules in order to monitor the production of amplification products during each cycle of the PCR reaction. Thus, the combination of excellent sensitivity and specificity, reproducible data, low contamination risk and reduced hand-on time, which make it a post-PCR analysis unnecessary, has made real-time PCR technology an appealing alternative to conventional PCR. The present paper attempts to provide a rigorous overview of fluorescent-based methods for nucleic acid analysis in real-time PCR described in the literature so far. Herein, different real-time PCR chemistries have been classified into two main groups; the first group comprises double-stranded DNA intercalating molecules, such as SYBR Green I and EvaGreen, whereas the second includes fluorophore-labeled oligonucleotides. The latter, in turn, has been divided into three subgroups according to the type of fluorescent molecules used in the PCR reaction: (i) primer-probes (Scorpions, Amplifluor, LUX, Cyclicons, Angler); (ii) probes; hydrolysis (TaqMan, MGB-TaqMan, Snake assay) and hybridization (Hybprobe or FRET, Molecular Beacons, HyBeacon, MGB-Pleiades, MGB-Eclipse, ResonSense, Yin-Yang or displacing); and (iii) analogues of nucleic acids (PNA, LNA, ZNA, non-natural bases: Plexor primer, Tiny-Molecular Beacon). In addition, structures, mechanisms of action, advantages and applications of such real-time PCR probes and analogues are depicted in this review.
Checklist for optimization and validation of real-time PCR assays.
Raymaekers M, Smets R, Maes B, Cartuyvels R.
J Clin Lab Anal. 2009 23(3): 145-151
Real-time polymerase chain reaction (PCR) is a frequently used technique in molecular diagnostics. To date, practical guidelines for the complete process of optimization and validation of commercial and in-house developed molecular diagnostic methods are scare. Therefore, we propose a practical guiding principle for the optimization and validation of real-time PCR assays. Based on literature, existing guidelines, and personal experience, we created a checklist that can be used in different steps of the development and validation process of commercial and in-house developed real-time PCR assays. Furthermore, determination of target values and reproducibility of internal quality controls are included, which allows a statistical follow-up of the performance of the assay. Recently, we used this checklist for the development of various qualitative and quantitative assays for microbiological and hematological applications, for which accreditation according to ISO 15189:2007 was obtained. In our experience, the use of the proposed guidelines leads to a more efficient and standardized optimization and validation. Ultimately, this results in reliable and robust molecular diagnostics. The proposed checklist is independent of environment, equipment, and specific applications and can be used in other laboratories. A worldwide consensus on this kind of checklist should be aimed at.
How good is a PCR efficiency estimate: Recommendations for precise and robust qPCR efficiency assessments
David Svec, Ales Tichopad, Vendula Novosadova, Michael W. Pfaffl, Mikael Kubistaa
Biomolecular Detection and Quantification; available online 11 March 2015
We have examined the imprecision in the estimation of PCR efficiency by means of standard curves based on strategic experimental design with large number of technical replicates. In particular, how robust this estimation is in terms of a commonly varying factors: the instrument used, the number of technical replicates performed and the effect of the volume transferred throughout the dilution series. We used six different qPCR instruments, we performed 1–16 qPCR replicates per concentration and we tested 2–10 μl volume of analyte transferred, respectively. We find that the estimated PCR efficiency varies significantly across different instruments. Using a Monte Carlo approach, we find the uncertainty in the PCR efficiency estimation may be as large as 42.5% (95% CI) if standard curve with only one qPCR replicate is used in 16 different plates. Based on our investigation we propose recommendations for the precise estimation of PCR efficiency: (1) one robust standard curve with at least 3–4 qPCR replicates at each concentration shall be generated, (2) the efficiency is instrument dependent, but reproducibly stable on one platform, and (3) using a larger volume when constructing serial dilution series reduces sampling error and enables calibration across a wider dynamic range.
Universal RNA reference materials for gene expression
Cronin M, Ghosh K, Sistare F, Quackenbush J, Vilker V, O'Connell C.
Clin Chem. 2004 50(8): 1464-1471
A workshop entitled "Metrology and Standards Needs for Gene Expression Technologies: Universal RNA Standards" was held in March 2003 to define the requirements for standardizing RNA-based molecular assays, specifically microarray and quantitative reverse-transcriptase-PCR technologies. NIST sponsored the workshop, and participants represented government, industry, academia, and clinic. Workshop participants concluded that as a first step, two RNA reference materials could be defined that would help in standardization of gene-expression technologies: an Assay Process Reference Material, and a Universal Array Hybridization Reference Material. The specific characteristics of these two standardized materials were broadly outlined. The Assay Process Material was proposed to be a pool of 96 expressed human sequences of defined composition, cloned in a defined vector and pooled in prescribed ways. The Universal Array Hybridization Material was defined as a pool of 12 "alien" synthetic sequences not expressed in any known genome to be used to control for variability in array hybridization methods. Work is underway at NIST and among members of the gene expression array community to further define these materials and make them available.
Correction of RT-qPCR data for genomic DNA-derived signals with ValidPrime.
Laurell H, Iacovoni JS, Abot A, Svec D, Maoret JJ, Arnal JF, Kubista M.
Inserm/Université Paul Sabatier UMR1048, Institut des Maladies Métaboliques et Cardiovasculaires (I2MC), BP84225, 31432 Toulouse cedex 4, France, Laboratory of Gene Expression, Institute of Biotechnology, Academy of Sciences of the Czech Republic, Prague, TATAA Biocenter AB, Göteborg, Sweden, Plateforme GeT (Génome et Transcriptome) du Génopole Toulouse, Toulouse, France and Faculté de Médecine, Université de Toulouse III and CHU de Toulouse.
Nucleic Acids Res. 2012 Jan 6.
Genomic DNA (gDNA) contamination is an inherent problem during RNA purification that can lead to non-specific amplification and aberrant results in reverse transcription quantitative PCR (RT-qPCR). Currently, there is no alternative to RT(-) controls to evaluate the impact of the gDNA background on RT-PCR data. We propose a novel method (ValidPrime) that is more accurate than traditional RT(-) controls to test qPCR assays with respect to their sensitivity toward gDNA. ValidPrime measures the gDNA contribution using an optimized gDNA-specific ValidPrime assay (VPA) and gDNA reference sample(s). The VPA, targeting a non-transcribed locus, is used to measure the gDNA contents in RT(+) samples and the gDNA reference is used to normalize for GOI-specific differences in gDNA sensitivity. We demonstrate that the RNA-derived component of the signal can be accurately estimated and deduced from the total signal. ValidPrime corrects with high precision for both exogenous (spiked) and endogenous gDNA, contributing ∼60% of the total signal, whereas substantially reducing the number of required qPCR control reactions. In conclusion, ValidPrime offers a cost-efficient alternative to RT(-) controls and accurately corrects for signals derived from gDNA in RT-qPCR.
Evaluation of external RNA controls for the standardisation of gene expression biomarker measurements.
Devonshire AS, Elaswarapu R, Foy CA
BMC Genomics. 2010 24;11: 662
BACKGROUND: Gene expression profiling is an important approach for detecting diagnostic and prognostic biomarkers, and predicting drug safety. The development of a wide range of technologies and platforms for measuring mRNA expression makes the evaluation and standardization of transcriptomic data problematic due to differences in protocols, data processing and analysis methods. Thus, universal RNA standards, such as those developed by the External RNA Controls Consortium (ERCC), are proposed to aid validation of research findings from diverse platforms such as microarrays and RT-qPCR, and play a role in quality control (QC) processes as transcriptomic profiling becomes more commonplace in the clinical setting.
RESULTS: Panels of ERCC RNA standards were constructed in order to test the utility of these reference materials (RMs) for performance characterization of two selected gene expression platforms, and for discrimination of biomarker profiles between groups. The linear range, limits of detection and reproducibility of microarray and RT-qPCR measurements were evaluated using panels of RNA standards. Transcripts of low abundance (≤ 10 copies/ng total RNA) showed more than double the technical variability compared to higher copy number transcripts on both platforms. Microarray profiling of two simulated 'normal' and 'disease' panels, each consisting of eight different RNA standards, yielded robust discrimination between the panels and between standards with varying fold change ratios, showing no systematic effects due to different labelling and hybridization runs. Also, comparison of microarray and RT-qPCR data for fold changes showed agreement for the two platforms.CONCLUSIONS: ERCC RNA standards provide a generic means of evaluating different aspects of platform performance, and can provide information on the technical variation associated with quantification of biomarkers expressed at different levels of physiological abundance. Distinct panels of standards serve as an ideal quality control tool kit for determining the accuracy of fold change cut-off threshold and the impact of experimentally-derived noise on the discrimination of normal and disease profiles.
Good laboratory practice when performing molecular amplification assays (QSOP 38)
Health Protection Agency (HPA), UK
Publication Type: Standard Operating Procedures, latest update 2010
National Standard Methods are a comprehensive referenced collection of clinical microbiology SOPs, algorithms (for virology and serology), and guidance notes, consisting of over 200 documents. National Standard Methods have been developed since 1996 by working groups of experienced laboratory-based medical and scientific microbiologists from throughout the HPA (previously the PHLS) and NHS.
The development of National Standard Methods and algorithms is undertaken under the auspices of the HPA in conjunction with the NHS and the National Public Health Service for Wales and with professional societies including the Association of Medical Microbiologists, Association of Clinical Microbiologists, Institute of Biomedical Science, Clinical Virology Network and the Scottish Microbiology Association.
Different real-time PCR systems yield different gene expression values.
Lu S, Smith AP, Moore D, Lee NM.
California Pacific Medical Center Research Institute, San Francisco, CA 94107, USA.
Mol Cell Probes. 2010 Oct;24(5): 315-320
Most polymerase chain reaction (PCR) systems employ pre-determined settings and proprietary master mixes that differ from one system to another. It is not known whether these differences may affect gene expression values. We compared two major real-time PCR technologies, from Life Technologies (formerly Applied Biosystems; ABI7500) and Roche Applied Science (LC480), using their default settings, proprietary reagents and other potential variables such as ramp rates and magnesium concentrations. We analyzed four genes (IL-8, COX2, ID-1 and CXCR2) in a human breast cancer cell line and found that two of them, though readily detected by ABI, were not detected using the Roche system. By altering some of the parameters and reagents used in the Roche protocol, we were able to detect expression of these two genes, but the level remained far below that detected by ABI, particularly for ID-1. When we tested three additional ID-1 primer pairs, two of these primer pairs yielded higher expression values in the LC system, yet still significantly lower than the values obtained in ABI. These results suggest critical differences in these two PCR systems, which could result in significant discrepancies in results reported by different laboratories.
An assessment of air as a source of DNA contamination encountered when performing PCR.
Witt N, Rodger G, Vandesompele J, Benes V, Zumla A, Rook GA, Huggett JF.
Centre for Infectious Diseases and International Health, Windeyer Institute for Medical Sciences, University College London, London, United Kingdom.
J Biomol Tech. 2009 20(5): 236-240
Sensitive molecular methods, such as the PCR, can detect low-level contamination, and careful technique is required to reduce the impact of contaminants. Yet, some assays that are designed to detect high copy-number target sequences appear to be impossible to perform without contamination, and frequently, personnel or laboratory environment are held responsible as the source. This complicates diagnostic and research analysis when using molecular methods. To investigate the air specifically as a source of contamination, which might occur during PCR setup, we exposed tubes of water to the air of a laboratory and clean hood for up to 24 h. To increase the chances of contamination, we also investigated a busy open-plan office in the same way. All of the experiments showed the presence of human and rodent DNA contamination. However, there was no accumulation of the contamination in any of the environments investigated, suggesting that the air was not the source of contamination. Even the air from a busy open-plan office was a poor source of contamination for all of the DNA sequences investigated (human, bacterial, fungal, and rodent). This demonstrates that the personnel and immediate laboratory environment are not necessarily to blame for the observed contamination.
Evaluation of different machines used to quantify genetic modification by real-time PCR.
Allnutt TR, Ayadi M, Berben G, Brodmann P, Lee D.
Food and Environmental Research Agency, Sand Hutton, York, YO411LZ, United Kingdom
J AOAC Int. 2010 93(4): 1243-1248.
Quantification of genetic modification (GM) is often undertaken to test for compliance with the European Union GM labeling threshold in food. Different control laboratories will often use common validated methods, but with different models of real-time PCR machines. We performed two separate ring trials to evaluate the relative precision and accuracy of different types of real-time PCR machines used to quantify the concentration of GM maize. Both trials used dual-labeled fluorogenic probes for quantification. The first ring trial used separate GM and reference assays (a single fluorescence channel), and the second used a combined duplex assay (two simultaneous fluorescence channels). Five manufacturers and seven models--including a 96-well microtiter-plate, rotary, and portable machines--were examined. In one trial, the machine used had a significant effect on precision, but in the other it did not. Overall, the degree of variation due to the machine model was lower than other factors. No significant repeatable difference in accuracy was observed between machine models. It was not possible to use sufficient replication of machine type in each laboratory to examine all sources of variation in this study, but the results strongly indicate that factors other than machine type or manufacturer (e.g., method or laboratory) contribute more to variation in a GM quantification result.
Using the Taguchi method for rapid quantitative PCR optimization with SYBR Green I.
Thanakiatkrai P, Welch L.
Centre for Forensic Science, University of Strathclyde, 204 George Street, Glasgow, G1 1XW, UK
Int J Legal Med. 2012 126(1): 161-165
Here, we applied the Taguchi method, an engineering optimization process, to successfully determine the optimal conditions for three SYBR Green I-based quantitative PCR assays. This method balanced the effects of all factors and their associated levels by using an orthogonal array rather than a factorial array. Instead of running 27 experiments with the conventional factorial method, the Taguchi method achieved the same optimal conditions using only nine experiments, saving valuable resources.
SYTO dyes and EvaGreen outperform SYBR Green in real-time PCR.
Division of Bioanalytical Chemistry, Office of Regulatory Science, Center for Food Safety and Applied Nutrition, U,S, Food and Drug Administration, 5100 Paint Branch Parkway, College Park, MD, USA 20740
BMC Res Notes. 2011 Jul 28;4: 263.
BACKGROUND: Real-time PCR can be carried out using either probes or DNA dyes. SYBR Green has been used the most, but it suffers from several drawbacks. Numerous other DNA dyes are commercially available, but with limited structural information. Dye behavior in real time PCR is difficult to predict, so empirical data are needed. In the work described here, a panel of 23 different DNA dyes--including green, orange, and red SYTO dyes, EvaGreen, and SYBR Green--were evaluated with respect to their performance in real time PCR.
FINDINGS: Data were analyzed for reaction inhibition, effects on amplicon melting temperature, fluorescent signal strength, and reaction efficiency. This is the first report of reaction efficiency using alternatives to SYBR Green. Results indicated substantial variation in performance even within the SYTO dye family. EvaGreen and the SYTO dyes 13, 16, 80, and 82 performed better than SYBR Green in general, and high reaction efficiencies can be achieved using these dyes.
CONCLUSIONS: Empirical data were generated for 23 DNA dyes. This paper confirms and extends previous findings that among commercially available DNA dyes, EvaGreen and certain SYTO dyes are the most desirable alternatives to the commonly used SYBR Green in real-time PCR.
Troubleshooting fine-tuning procedures for qPCR system design
Raso A, Mascelli S, Nozza P, Ugolotti E, Vanni I, Capra V, Biassoni R.
Neurosurgery Unit, Giannina Gaslini Children's Research Hospital, Genoa, Italy
J Clin Lab Anal. 2011 25(6): 389-394
Quantitative real-time PCR (qPCR) has been improved and optimized over the past decade for a wide range of applications. Design of primers and probes is one of the crucial steps to obtain high system efficiency of qPCR since design pitfalls influence negatively amplification performances. We report the results of some experiments. First, we demonstrate the utility of optimal primer design and concentration in PCR by constructing suboptimal primers, for instance with hairpin and primer-dimers secondarystructures, and quantifying the decrease in efficiency of amplification. Second, we show the adverse effects of the target sequence harboring stable secondary structures on the primer binding sites. Finally, we let see that the mere use of probe-based detection is not enough to ensure robustness of qPCR data, because the eventual detrimental products generated by primers not well designed may influence in any case the PCR efficiency.