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 the latest publications  real-time PCR optimisation (5)

Eleven golden rules of quantitative RT-PCR    ... NEW !

The MIQE guidlines  &  The MIQE press review   ...UPDATE !

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.

Current problems in quantitative real-time RT-PCR
by T. Nolan, RE. Hands & SA Bustin;  Nature Protocols (2006) Vol. 1, No. 3; p1559-1582

The real-time reverse transcription polymerase chain reaction (RT-qPCR) addresses the evident requirement for quantitative data analysis in molecular medicine, biotechnology, microbiology and diagnostics and has become the method of choice for the quantification of mRNA. Although it is often described as a ‘‘gold’’ standard, it is far from being a standard assay. The significant problems caused by variability of RNA templates, assay designs and protocols, as well as inappropriate data normalization and inconsistent data analysis, are widely known but also widely disregarded. The widespread use of this technology has resulted in the development of numerous protocols that generate quantitative data using:
  • fresh, frozen or archival FFPE (formalin-fixed, paraffinembedded) samples,
  • whole-tissue biopsies, microdissected samples, single cells, tissue culture cells,
  • total or mRNA,
  • a range of different cDNA priming strategies,
  • different enzymes or enzyme combinations,
  • assays of variable efficiency, sensitivity and robustness,
  • diverse detection chemistries, reaction conditions, thermal cyclers and
  • individual analysis and reporting methods.
This obvious lack of standardization at every step of the assay (Figure 1) is exacerbated by significant differences in sample processing, use of controls, normalization methods and quality control management and has serious implications for the reliability, relevance and reproducibility of RT-qPCR. An overview of the considerations relating to procedures and alternative steps for carrying out the RT-qPCR reaction is shown in Figure 2.

Click to enlarge!

Quantification of mRNA using real-time RT-PCR

Tania Nolan, Rebecca E Hands & Stephen A Bustin
Nature Protocols (2006) Vol. 1, No. 3; p1559-1582

PCR Troubleshooting and Optimization - The Essentail Guide
edited by Suzanne Kennedy & Nick Oswald

Chapter 5 - RT-PCR Optimisation Strategies
by Martina Reiter & Michael W. Pfaffl
Physiology Weihenstephan, Weihenstephaner Berg 3, 85354 Freising, Germany

PCR technology is based on a simple principle; an enzymatic reaction that increases the initial amount of nucleic acids. This method makes it possible to detect specific mRNA transcripts in any biological sample. Performing RT-PCR analysis does not only comprehend this experimental PCR step. Following the whole workflow of a RT-PCR quantitative analysis, it starts with the sampling step, followed by nucleic acid extraction and stabilization, cDNA synthesis and finally the qPCR where the mRNA quantification takes place. Problems arise when optimization of the experimental work flow becomes necessary because of high technical variations. The PCR reaction itself is a quite stable reaction with reproducibility between 2-8%. Therefore the source of experimental variances can often be found in the pre-PCR analytical steps. Usually this is neglected and optimization is done for PCR reaction only. In this chapter – RT-PCR optimization strategies - the whole workflow of RT-PCR experiment will be discussed, because the identification of the source of variability is only possible following error accumulation in every single step. Reliable data can be created when the technical variance caused by the experimental steps is kept as low as possible. In this chapter many recommendations to decrease the technical variance can be found.

PCR was invented over 25 years ago by Kary Mullis [Saiki, 1985], for which he received the Nobel Prize in chemistry in 1993 [Malmström, 1997]. PCR is considered to be the innovation which allowed molecular biology to evolve to the current level. It has become an indispensable technique in life science research and more recently in routine human and veterinary diagnostics. PCR has evolved over the past decades from a technically complicated method to a simple and easy to apply method. There is a wide variety of ready-to-use reagents available that allows those with some basic training and who master the skill of pipetting to perform a PCR. Enzymes and instruments have been continously engineered to speed up the PCR process, so that a PCR can presently be performed in less than half an hour. However, the simplicity of the method is its strength and weakness at the same time. As it is relatively easy to generate a result many PCR users fail to appreciate the quality control that is required to generate reliable and meaningful results. With the more recent use of PCR in diagnostics the call for quality control is increasing in accredited and quality aware laboratories. An increasing number of laboratories either elect or are required to obtain an ISO 17025 [CEN, 2005] or ISO 15189 [CEN, 2007] accreditation to guarantee the quality of the results generated. At the same time, the research community’s call for biologically meaningful conclusions is increasing in parallel. In 2009 a group of leading PCR scientists published guidelines (MIQE) [Bustin, 2009], that assist qPCR users to design a robust qPCR experiment that leads to trustworthy and biologically meaningful results which can be reproduced in any other laboratory. The main variables of the (q)PCR reaction are the purity and quality of the template DNA or cDNA, the design, purity and concentration of the primers and probes, the concentration of the different reagents, the type of buffer and the type of enzyme, the tubes, strips or plates and the thermocycler used (figure 1). The vast majority of (q)PCR optimizations are performed on the variables of DNA, primers and template. Yet, very little attention is paid to the contribution of the variability of tubes and thermocycler to the (q)PCR result, as they are incorrectly considered to be constants rather than variables.

Figure 1:   Main variables of qPCR process

The goal of this Thermocycler Calibration Guide is to illustrate which types of thermocycler variability do exist, show the impact of thermocycler variability on the outcome of PCRs or qPCRs, and offer practical solutions how to eliminate or control thermocycler variability. The practical protocols allows us to put into practical use the data from CYCLERtest Calibration Certificates and Reports. Examples will be given showing how thermocyclers can be aligned and programmed to mimic each other. Furthermore, examples will be given showing how calibration results can be used for validation purposes when working under ISO 17025, ISO 15189 accreditation and many other regulations. This guide will allow PCR and qPCR users to explore the full potential of CYCLERtest thermocycler calibration data.

download => Cyclertest thermocycler calibration guide

The below publications, protocols, and international programs should help, to optimize your PCR and qRT-PCR assays:

REVIEW - qPCR Satellite Symposium
Optimisation,  Normalisation  &  Standardisation
10-11th March 2005

Real-Time PCR Troubleshooting Tool

Having problems with your gene expression or SNP genotyping experiments? Do your amplification curves look sigmoidal, or do you have no curves at all? Do your allelic discrimination plots have diffuse or trailing clusters? Our interactive troubleshooting tool will guide you step by step to a solution.

International Programs:

The MIQE Guidelines Minimum Information for Publication of Quantitative Real-Time PCR Experiments

Stephen A. Bustin, Vladimir Benes, Jeremy A. Garson, Jan Hellemans, Jim Huggett, Mikael Kubista, Reinhold Mueller, Tania Nolan, Michael W. Pfaffl, Gregory L. Shipley, Jo Vandesompele, & Carl T. Wittwer
Clinical Chemistry 2009, 55(4): 611-622

BACKGROUND:  Currently, a lack of consensus exists on how best to perform and interpret quantitative real-time PCR (qPCR) experiments.  The problem is exacerbated by a lack of sufficient experimental detail in many publications, which impedes a reader's ability to evaluate critically the quality of the results presented or to repeat the experiments.
CONTENT:  The Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines target the reliability of results to help ensure the integrity of the scientific literature, promote consistency between laboratories, and increase experimental transparency. MIQE is a set of guidelines that describe the minimum information necessary for evaluating qPCR experiments. Included is a checklist to accompany the initial submission of a manuscript to the publisher. By providing all relevant experimental conditions and assay characteristics, reviewers can assess the validity of the protocols used. Full disclosure of all reagents, sequences, and analysis methods is necessary to enable other investigators to reproduce results. MIQE details should be published either in abbreviated form or as an online supplement.
SUMMARY:  Following these guidelines will encourage better experimental practice, allowing more reliable and unequivocal interpretation of qPCR results.

Nucleic Acids Research Group (NARG)
The Nucleic Acid Research Group (NARG) of the Association of Biomolecular Resource Facilities (ABRF) qPCR survey. The aim of the survey was to determine the current status of real-time PCR technology in laboratories around the world, particularly core laboratories. Your answers will help us "take the pulse" of the real-time qPCR community  =>

Mission Statement
  • To facilitate the use of nucleic acid-based technologies in research
  • To examine current trends of nucleic acid-based technologies
  • To assist member laboratories in self-evaluation and growth
  • To promote, encourage, and reward excellence in member laboratories
    NARG 2009 study: Priming strategies for cDNA Synthesis
        - View ABRF2009 presentation
        - View ABRF2009 presentation on routine RNA handling
    2) NARG 2008 study: Priming Strategies for cDNA Synthesis
        - View ABRF2008 NARG presentation
        - View ABRF2008 Poster (434K)
    3) NARG 2007 Real-Time PCR Survey The Nucleic Acid Research Group (NARG) of the Association of Biomolecular Resource Facilities (ABRF) invites anyone who uses “Real-Time” quantitative PCR (qPCR) to participate in our on-line survey. The aim of the survey is to determine the current status of real-time PCR technology in laboratories around the world, particularly core laboratories. Your answers will help us "take the pulse" of the real-time qPCR community. Submissions are anonymous and results will be freely available via a "web poster". This survey will be “open” until February 2, 2007. Results will be presented at the ABRF 2007 annual meeting in Tampa Bay, FL, Mar 31-Apr 3, 2007 and will be available "on line" by May 1, 2007. We think it will be worth your time to participate in this study.
        - View_2007_Survey_Presentation
        - View_2007_Survey_Poster
        - View_2007_Survey_Questions
        - View_2007_Survey_RawData
    4) NARG 2006 Study: Priming Strategies for Real-time RT-PCR The purpose of this study is to provide an opportunity for participating laboratories to gain crucial information about the variability of the RT-step of the qPCR assay and about the comparability of qPCR results obtained using different cDNA priming strategies. In addition, the study will act as an audit for participating laboratories, who will be able to compare the results from their protocols, techniques and equipment with those from other laboratories around the world. The study is open for those who use Taqman® probe-based or SYBR Green I-based assay systems. Deadline for sample submission is Dec. 15, 2005. Data will be presented at the ABRF 2006 annual meeting in Long Beach, CA, February 11 - 14, 2006. We think it will be worth your time to participate in this study.
        - View Study announcement (pdf) (46K)
        - ViewStudy information(pdf)
        - View Study Questions(pdf) (124K)
        - View Study Examples (xls) (68K)
        - View Slides of RG Presentation on ABRF 2006 Study (9,505K)
        - View Slides of Multiplex Development/Optimization
    5) NARG 2005 Study: Validation of Your Reverse Transcription Real-Time PCR Technique The purpose of this study is to give investigators an opportunity to test their reverse transcription real-time PCR technique and to gather information about the performance of various platforms, variations due to reagents and how people analyze their data. The study is open for those who use both Taqman® type systems and SYBRgreen systems. Deadline for sample submission is Dec. 15, 2004. A poster containing a table showing (anonymously) how the individual participants fared in their assay efforts will be posted on this page. Data will be presented at the ABRF 2005 annual meeting in Savannah, GA, March 5 - March 8, 2005.
        - View Study Invitation (pdf) (88K)
        - View Study Information (pdf) (96K)
        - View Submission Instructions (pdf) (54K)
        - View Poster of preliminary results
        - View Slides of Research Group Presentation
        - View Study Questions (218K)
    6) NARG 2003/2004 Real-Time PCR Survey

    This survey was designed to determine the current status of real-time PCR technology in laboratories around the world, especially Core laboratories. The answers allowed us to "take the pulse" of the real-time PCR community. The survey was open from November 15, 2003 until January 15, 2004. Submissions were anonymous . Results were presented at the ABRF 2004 annual meeting in Portland, OR, Feb 28-Mar 2, 2004 and at the Ist International qPCR symposium 3rd - 6th March, 2004 in Freising-Weihenstephan, Germany. Results are available below in several forms. All material is copyrighted and for scientific use only.

  • Raw data: This is a PDF file of the raw data. Please be aware that there are two duplicate entries and 3 null entries in this data.
  • Web poster: A JPG file summarizing the survey data.
  • Research Group presentation from ABRF 2004 by Brian Holloway and Tony Yeung

  •     - View survey questions (53K)
        - View raw data (4,082K)
        - View Poster (804K)
        - View Research Group presentation (312K)
    7) NARG 2004 Taqman Primer/Probe Design Study The purpose of this study was to give investigators an opportunity to design an optimal set of primers/probe for a common gene and have them tested empirically for effectiveness. We were able to demonstrate some of the basic principles of Taqman Assay design. A table showing how the individual participants fared in their design efforts is posted below. Final analysis of results will be published in a peer reviewed journal. A copy of the poster presented at the ABRF 2004 annual meeting in Portland, OR, Feb 28-Mar 2, 2004 and at the Ist International qPCR symposium 3rd - 6th March, 2004 in Freising-Weihenstephan, Germany may be obtained by contacting
        - View study invitation (53K)
        - IFNg CDS (11K)
        - Primer 3 web site
        - Suggestions for assay design/Primer 3 use (352K)
        - Participant Results (47K)
        - View Study Survey Questions (159K)
        - View Poster of preliminary results (258K)
    8) NARG 2003 Study Announcement

    Real-time PCR technology is of increasing importance in research. The commercial cost of dual-labeled probes for real-time PCR reactions is high because of traditional HPLC or gel purification steps. NARG believed that these probes may be used without purification, if carefully prepared, thereby reducing the cost and making the synthesis of real-time PCR probes feasible and practical for any DNA synthesis laboratory.

    NARG proposed to test whether all ABRF DNA synthesis labs are able to make quality dual-labeled probes suitable for real-time PCR reactions without requiring purification. It also wanted to determine the conditions under which highly purified probes may excel over unpurified probes.
        - View Study announcement (pdf) (13K)
        - View Recommended Protocols for Synthesis (pdf)
        - View Sample survey (pdf) (188K)
        - View Examples of Quality Analysis (pdf)
        - View Tutorial on Probe Synthesis (pdf)

    9) NARG 2003 Results
        - View Summary of Results (12K)
        - View PAGE data (108K)
        - View CE data (943K)
        - View DHPLC data (3,785K)
        - View Taqman® data (3,458K)
        - View 2003 ABRF Poster
        - Go to Biotechniques Publication Information
    10) NARG 2001 Research Group Presentation
        - View slides (1,391K)
    11) Previous NARG studies
        - NARG data repository

    Eleven golden rules of quantitative RT-PCR

    Eleven golden rules of quantitative RT-PCR

    Udvardi MK, Czechowski T, Scheible WR.
    Plant Cell. 2008 20(7): 1727

    Reverse transcription followed by quantitative polymerase chain reaction analysis, or qRT-PCR, is an extremely sensitive, cost-effective method for quantifying gene transcripts from plant cells. The availability of nonspecific double-stranded DNA (dsDNA) binding fluorophors, such as SYBR Green, and 384-well-plate real-time PCR machines that can measure fluorescence at the end of each PCR cycle make it possible to perform qRT-PCR on hundreds of genes or treatments in parallel. This has facilitated the comparative analysis of all members of large gene families, such as transcription factor genes (Czechowski et al., 2004). Given the relatively low cost of PCR reagents, and the precision, sensitivity, flexibility, and scalability of qRT-PCR, it is little wonder that thousands of research labs around the world have embraced it as the method of choice for measuring transcript levels. However, despite its popularity, we continue to see systematic errors in the application of methods for qRT-PCR analysis, which can compromise the interpretation of results. The letter to the editor by Gutierrez et al. in this issue highlights one of many common sources of error, namely, the inappropriate choice of reference genes for normalizing transcript levels of test genes prior to comparative analysis of different biological samples. The following are 11 golden rules of qRT-PCR that, when observed, should ensure reproducible and accurate measurements of transcript abundance in plant and other cells. These rules are for relative quantification of RNA using two-step RT-PCR (where the product of a single RT reaction is used as template in multiple PCR reactions), SYBR Green to detect gene-specific PCR products, and reference genes for normalizing transcript levels of test genes before comparing samples. Further details can be found elsewhere (Czechowski et al., 2004, 2005). Most of these rules also apply to relative quantification methods that employ sequence-specific fluorescent probes, such as TaqMan probes, and to absolute quantification methods.

    1. Harvest material from at least three biological replicates to facilitate statistical analysis of data, freeze immediately in liquid nitrogen, and store at –80°C to preserve full-length RNA.
    2. Use an RNA isolation procedure that produces high-quality total RNA from all samples to be analyzed. Check RNA quality using an Agilent 2100 Bioanalyzer (RNA integrity number, RIN > 7 and ideally > 9) or by electrophoretic separation on a high-resolution agarose gel (look for sharp ethidium bromide–stained rRNA bands) and spectrophotometry (A260/A280 > 1.8 and A260/A230 > 2.0). Quantify RNA using A260 values.
    3. Digest purified RNA with DNase I to remove contaminating genomic DNA, which can act as template during PCR and lead to spurious results. Subsequently, perform PCR on the treated RNA, using gene-specific primers, to confirm absence of genomic DNA.
    4. Perform RT reactions with a robust reverse transcriptase with no RNaseH activity (like SuperScriptIII from Invitrogen or ArrayScript from Ambion) to maximize cDNA length and yield. Use ultraclean oligo(dT) primer of high integrity. qRT-PCR gene expression measurements are comparable only when the same priming strategy and reaction conditions are used in all experiments and reactions contain the same total amount of RNA (Ståhlberg et al., 2004).
    5. Test cDNA yield and quality. Perform qPCR on an aliquot of cDNA from each sample, using primers to one or more reference genes that are known to be stably expressed in the organ(s) / tissue(s) under the range of experimental conditions tested. Threshold cycle (Ct) values should be within the range mean ±1 for each reference gene across all samples to ensure similar cDNA yield from each RT reaction. Quality of cDNA can be assessed using two pairs of primers for a reference gene that are ~1 kb apart. Typically, the Ct value for the primer pair at the 5'-end of a cDNA will be higher than the Ct value of the primer pair at the 3'-end, as reverse transcription begins at the 3' [poly(A)] end of the template mRNA and does not always extend to the 5'-end of the template. Ideally, the Ct value of the 5'-end primer pair should not exceed that of the 3'-end pair by more than one cycle number.
    6. Design gene-specific PCR primers using a standard set of design criteria (e.g., primer Tm = 60 ± 1°C, length 18 to 25 bases, GC content between 40 and 60%), which generate a unique, short PCR product (between 60 and 150 bp) of the expected length and sequence from a complex cDNA sample in preliminary tests, to facilitate multiparallel qPCR using a standard PCR program. The 3'-untranslated region is a good target for primer design because it is generally more unique than coding sequence and closer to the RT start site.
    7. Reduce technical errors in PCR reaction setup by standardizing (robotize if possible) and minimizing the number of pipetting steps. Mix cDNA with qPCR reagents, then aliquot a standard volume of this "master mix" into each reaction well containing a standard volume of specific primers. Set up reactions in a clean environment free of dust, preferably under a positive airflow hood. Routinely check for DNA contamination of primer and reagent stocks by performing PCR reactions on no template (water) controls.
    8. For relative quantification of transcript levels, design and test gene-specific primers for at least four potential reference genes selected from the literature (e.g., Czechowski et al., 2005) or from your own experience that are likely to be stably expressed throughout all organs and treatments to be compared. Validate reference genes in preliminary experiments on the range of tissues and treatments you wish to compare using a foreign cRNA added to each RNA sample prior to RT-PCR to normalize data for reference gene transcripts prior to assessment of their expression stability (Czechowski et al., 2005).
    9. Perform real-time PCR on test and reference genes in parallel for each sample to capture fluorescence data on dsDNA after each cycle of amplification. Also, perform dsDNA melting curve analysis at the end of the PCR run. When relying on nonspecific DNA binding fluorophors, such as SYBR Green, to quantify relative dsDNA amount, ensure that only a single PCR amplicon of the expected length and melting temperature is produced using gel electrophoresis and PCR amplicon melting curve data, respectively. We typically use a commercial mixture of hot-start Taq polymerase, SYBR Green, and other reagents, such as Power SYBR Green Master Mix from Applied Biosystems, and have observed significant differences in the efficacy (PCR efficiency, specificity, and/or yield) of such products from different suppliers.
    10. Determine which reference gene(s) is best for normalization of test gene transcript levels amongst all samples (e.g., using geNorm [Vandesompele et al., 2002] or BestKeeper software [Pfaffl et al., 2004]), which use as input not only the Ct value, but also the PCR efficiency for each reaction. PCR efficiency can be derived conveniently from amplification plots using the program LinRegPCR (Ramakers et al., 2003). Estimation via the classical calibration dilution curve and slope calculation is also possible, albeit more complicated.
    11. Finally, calculate relative transcript abundance for each gene in each sample using a formula that incorporates PCR efficiency for the test gene and Ct values for both test and reference genes.

      New papers for optimising your qPCR:

    Recent papers for optimising your qPCR:

    PCR Additives

    A variety of PCR additives and enhancing agents have been used to increase the yield, specificity and consistency of PCR reactions. Whilst these additives may have beneficial effects on some amplifications it is impossible to predict which agents will be useful in a particular context and therefore they must be empirically tested for each combination of template and primers. Some of the more popular of these additives are  listed in the table below along with references describing their use.



    (dimethyl sulfoxide)

    Amplifications 5: 16

    Gene 140: 1
    Nucleic Acids Research 18: 1666

    = [carboxymethyl]trimethylammonium)

    Biochemistry 32: 137
    BioTechniques 21: 1102
    Genome Research 6: 633
    Nucleic Acids Research 25: 3957
    Proceedings of the
    National Academy of Sciences of the United States of America70: 298
    Trends in Biochemical Science 22: 225


    Nucleic Acids Research 18: 7465

    Non-ionic detergents
    e.g. Triton X-100, Tween 20 or Nonidet P-40 (NP-40)

    Biotechniques 12: 332
    Nucleic Acids Research 18: 1309

    (tetramethylammonium chloride)

    Nucleic Acids Research 18: 4953
    Nucleic Acids Research 23: 3343


    Nucleic Acids Research 16: 3360

    (bovine serum albumin)

    Applied and environmental microbiology 62:1102-1106
    BioTechniques 23:504
    BioTechniques 25:564
    Nucleic Acids Research 16: 9775

    T4 gene 32 protein

    Applied and Environmental Microbiology 62:1102-1106

    DMSO at 2-10% may be necessary for amplification of some templates, however 10% DMSO can reduce Taq polymerase activity by up to 50% (Gelfand 1989) so it should not be used routinely. DMSO is thought to reduce secondary structure and is particularly useful for GC rich templates.

    A number of PCR additives are now comercially available, however the identities of these agents are not usually revealed by their suppliers. Frackman et al.(1998) have demonstrated (using NMR analysis) that the PCR additive provided by QIAGEN in their PCR core kit (Q-Solution) and that provided by CLONTECH in the Advantage-GC cDNA PCR kit is in fact Betaine which is available at a fraction of the cost as a 5M solution from Sigma-Aldrich (cat. # B 0300), but be sure to use Betaine or Betaine (mono)hydrate and not Betaine HCl. Other products suspected of consisting largely of Betaine include the "GC-RICH solution enhancer" from Roche, "TaqMaster enhancer" from Eppendorf, "GC-melt" from Clontech and "FailSafe enhancer" (formerly "MasterAmp PCR Enhancment Technology") from Epicentre (Weissensteiner, pers. comm.). Betaine is generally used at a final concentration of 1.0-1.7M.

    Formamide is generally used at 1-5% and 10% formamide is reported (Gelfand 1989) to have no effect on the activity of Taq polymerase, however, Sarkar et al. (1990) (see table for ref.) found that 1.25% formamide worked as well as 2.5% and 5%, and no amplification was seen at 10% so it seems prudent not to use concentrations of formamide greater than strictly necessary for optimal amplification.

    Non-ionic detergents stabilise Taq polymerase and may also supress the formation of secondary structure. 0.1-1% Triton X-100, Tween 20 or NP-40 may increase yield but may also increase non-specific amplification. As little as 0.01% SDS contamination of the template DNA (left-over from the extraction procedure) can inhibit PCR by reducing Taq polymerase activity to as low as 10%, however, inclusion of 0.5% Tween-20 or -40 will effectively neutralise this effect (Gelfand 1989).

    TMAC is generally used at a final concentration of 15-100mM to eliminate non-specific priming. TMAC has is also used to reduce potential DNA-RNA mismatch (Proceedings of the National Academy of Sciences of the United States of America 82: 1585) and improve the stringency of hybridization reactions (Nucleic Acids Research 16: 4637).

    The base analogue 7-deaza-2'-deoxyguanosine may facilitate amplification of templates with stable secondary structures when used in place of dGTP in a ratio of 3: 1, 7-deaza-2'-deoxyguanosine: dGTP.

    BSA has proven particularly useful when attempting to amplify ancient DNA or templates which contain PCR inhibitors such as melanin.


    Frackman, S., Kobs, G., Simpson, D. and Storts, D. 1998. Betaine and DMSO: enhancing agents for PCR. Promega Notes 65: 27.
    Gelfand, D. H. 1988.
    In Erlich, H. A. (ed.) PCR Technology. p.17. Stockton Press, NY.

    Novel UNIQ qPCR PT Scheme

    A new PT scheme has been developed, providing an opportunity for users to obtain confidential and unbiased assessment of their QPCR performance.  The PT has been developed under the DTI-funded Measurements for Biotechnology (MfB) programme as part of an initiative to improve the comparability of results between laboratories, and the materials for the first round will be sent out this September.
    The scheme takes an innovative approach, and uses synthetic DNA targets in a proprietary artificial matrix to allow researchers and analysts from all sectors to participate without fears of laboratory contamination.  Participants will be required to perform basic DNA quantification of a high concentration DNA stock, DNA extraction of 9 unknown samples and QPCR analysis of the 9 unknown samples plus 3 additional unknowns. Results will be compared between participating laboratories, and performance will be scored using a conventional PT Z-scoring approach. By taking part in the scheme, participants will be able to demonstrate the overall effectiveness of their performance, which is increasingly important to maintain the confidence of customers and funding bodies.               UNIQ-PT-scheme-flier.pdf

    A form to register interest can be downloaded here:   mfb-application-form.doc

      Steps and variables of a successful mRNA quantification using real-time RT-PCR

    Click to enlarge ! Click to enlarge !