Steps and variables of a successful mRNA quantification using real-time RT-PCR
Recent papers for optimising your qPCR:
Standardizing the standards.
Evaluation of probe chemistries and platforms to improve the detection limit of real-time PCR.
Reynisson E, Josefsen MH, Krause M, Hoorfar J.
J Microbiol Methods. 2006 Aug;66(2):206-16. Epub 2005 Dec 20.
A validated PCR-based Salmonella method targeting a 94-bp sequence of the ttr gene was used as a model to compare six different combinations of reporter and quencher dyes of a TaqMan probe, on three different instruments, to improve the detection limit in a real-time PCR assay with the aim of a same-day analysis. The use of locked nucleic acids (LNA) and Scorpion probes were also tested. The combination FAM-BHQ1 or Cy5-BHQ3, both dark quenchers, gave the best results (Cycle threshold (Ct) of 25.42+/-0.65 and 24.47+/-0.18 at 10(3) DNA copies). When comparing different probe technologies, the LNA probe (FAM-BHQ1) was the most sensitive with the strongest fluorescence signal (dR last 48066), resulting in 0.6 to 1.1 lower Ct values than a DNA TaqMan probe, and 1.9 to 4.0 lower Ct than the Scorpion system (FAM-BHQ1). The RotorGene real-time PCR instrument gave 0.4-1.0 lower Ct values (more sensitive) than the Mx3005p, and 1.5-3.0 lower than the ABI 7700. Using the LNA in a RotorGene instrument, we detected the following Salmonella DNA copies in 1-ml pre-enriched samples: fishmeal (100 copies), chicken rinse (100 copies) and pig feces (10 copies). The detection probability of the final assay on inoculated fecal samples was 100% at 2x10(4) copies per ml. In conclusion, the LNA probe with annealing temperature of 65 degrees C could be useful for more sensitive detection limits.
Optimized real-time quantitative PCR measurement of male fetal DNA in maternal plasma.
Zimmermann B, El-Sheikhah A, Nicolaides K, Holzgreve W, Hahn S.
Clin Chem. 2005 Sep;51(9): 1598-604. Epub 2005 Jul 14.
University Women's Hospital/Department of Research, University Hospital Basel, Switzerland.
BACKGROUND: Circulating fetal DNA (cfDNA) in maternal plasma has been measured to investigate its possible relationship with pregnancy-related disorders, including fetal trisomy 21 and preeclampsia. The circulating concentrations of single-copy fetal genes, however, are close to the detection limits of PCR methods.
METHODS: We optimized a protocol for the real-time quantitative PCR amplification of the multicopy sequence DYS14 on the Y-chromosome. This was compared with an established real-time PCR assay for the single-copy SRY gene.
RESULTS: By probit regression analysis, the measurements of male DNA by the DYS14 assay had a 10-fold lower detection limit (0.4 genome equivalents) than did measurements of SRY. For plasma samples from women in the first trimester of pregnancy, imprecision (CV) was 2%-22% when amplifying DYS14 compared with 26%-140% for SRY.
CONCLUSIONS: The low copy numbers of fetal DNA in plasma of women in the first trimester of pregnancy cannot be measured precisely when targeting single-copy sequences. Better results are obtained by amplifying a sequence that is present in multiple copies per male genome.
Evaluation of dual-labeled fluorescent DNA probe purity versus performance in real-time PCR.
Yeung AT, Holloway BP, Adams PS, Shipley GL.
Biotechniques. 2004 Feb;36(2):266-70, 272, 274-5.
Fox Chase Cancer Center, Philadelphia, PA, USA.
Real-time PCR technology using dual-labeled fluorescent oligonucleotide probes allows for sensitive, specific, and quantitative determination of mRNA or DNA targets. Historically, dual-labeled probes have been the most expensive reagent in real-time PCR because of the postsynthesis high-performance liquid chromatography (HPLC) and/or gel purification steps required due to limitations in traditional synthesis chemistry. The recent availability of quencher reagents that allow the 3' quencher incorporation as part of the on-machine synthesis has presented the possibility that probes, when carefully synthesized, may be used without extensive postsynthesis purification. This would substantially reduce cost, making the synthesis of dual-labeled fluorescent probes affordable to any DNA synthesis laboratory. The Nucleic Acids Research Group (NARG) of the Association of Biomolecular Resource Facilities (ABRF) (Santa Fe, NM, USA) tested the hypothesis that now any DNA synthesis laboratory is capable of making quality dual-labeled fluorescent probes suitable for real-time PCRs without the need for postsynthesis purification. Members of the DNA synthesis community synthesized dual-labeled human beta-actin probes and submitted them for quality and functional analysis. We found that probes that were at least 20% pure had the same efficiency as those near 100% purity, but the sensitivity of the assay was reduced as the level of purity decreased.
Determination of allele frequency in pooled DNA: comparison of three PCR-based methods.
Wilkening S, Hemminki K, Thirumaran RK, Bermejo JL, Bonn S, Forsti A, Kumar R.
Biotechniques. 2005 Dec;39(6):853-8.
German Cancer Research Center, Molecular Genetic Epidemiology, Heidelberg, Germany.
Determination of allele frequency in pooled DNA samples is a powerful and efficient tool for large-scale association studies. In this study, we tested and compared three PCR-based methods for accuracy, reproducibility, cost, and convenience. The methods compared were: (i) real-time PCR with allele-specific primers, (ii) real-time PCR with allele-specific TaqMan probes, and (iii) quantitative sequencing. Allele frequencies of three single nucleotide polymorphisms in three different genes were estimated from pooled DNA. The pools were made of genomic DNA samples from 96 cases with basal cell carcinoma of the skin and 96 healthy controls with known genotypes. In this study, the allele frequency estimation made by real-time PCR with allele-specific primers had the smallest median deviation (MD) from the real allele frequency with 1.12% (absolute percentage points) and was also the cheapest method. However; this method required the most time for optimization and showed the highest variation between replicates (SD = 6.47%). Quantitative sequencing, the simplest method, was found to have intermediate accuracies (MD = 1.44%, SD = 4.2%). Real-time PCR with TaqMan probes, a convenient but very expensive method, had an MD of 1.47%and the lowest variation between replicates (SD = 3.18%).
EQUAL-quant: an international external quality assessment scheme for real-time PCR.
BACKGROUND: Quantitative gene expression analysis by real-time PCR is important in several diagnostic areas, such as the detection of minimum residual disease in leukemia and the prognostic assessment of cancer patients. To address quality assurance in this technically challenging area, the European Union (EU) has funded the EQUAL project to develop methodologic external quality assessment (EQA) relevant to diagnostic and research laboratories among the EU member states. We report here the results of the EQUAL-quant program, which assesses standards in the use of TaqMan probes, one of the most widely used assays in the implementation of real-time PCR.
METHODS: The EQUAL-quant reagent set was developed to assess the technical execution of a standard TaqMan assay, including RNA extraction, reverse transcription, and real-time PCR quantification of target DNA copy number.
RESULTS: The multidisciplinary EQA scheme included 137 participating laboratories from 29 countries. We demonstrated significant differences in performance among laboratories, with 20% of laboratories reporting at least one result lacking in precision and/or accuracy according to the statistical procedures described. No differences in performance were observed for the >10 different testing platforms used by the study participants.
CONCLUSIONS: This EQA scheme demonstrated both the requirement and demand for external assessment of technical standards in real-time PCR. The reagent design and the statistical tools developed within this project will provide a benchmark for defining acceptable working standards in this emerging technology.
Diagnostic PCR: validation and sample preparation are two sides of the same coin.
Increased use of powerful PCR technology for the routine detection of pathogens has focused attention on the need for international validation and preparation of official non-commercial guidelines.Bacteria of epidemiological importance should be the prime focus, although a "validation infrastructure" once established could easily be adapted for PCR-based detection of viruses and parasites. The aim of standardization should be the widespread adoption of diagnostic PCR for routine pathogen testing. European experience provides the impetus for realization of this vision through preparation of quantitative reference DNA material and reagents, production of stringent protocols and tools for thermal cycler performance testing, uncomplicated sample preparation techniques, and extensive ring trials for assessment of the efficacy of selected matrix/pathogen detection protocols.
due to detection of DNA originating from dead cells.
Wolffs P, Norling B, Radstrom P.
J Microbiol Methods. 2005 Mar;60(3):315-23.
Applied Microbiology, Lund Institute of Technology, Lund University, SE-221 00 Lund, Sweden.
Real-time PCR technology is increasingly used for detection and quantification of pathogens in food samples. A main disadvantage of nucleic acid detection is the inability to distinguish between signals originating from viable cells and DNA released from dead cells. In order to gain knowledge concerning risks of false-positive results due to detection of DNA originating from dead cells, quantitative PCR (qPCR) was used to investigate the degradation kinetics of free DNA in four types of meat samples. Results showed that the fastest degradation rate was observed (1 log unit per 0.5 h) in chicken homogenate, whereas the slowest rate was observed in pork rinse (1 log unit per 120.5 h). Overall results indicated that degradation occurred faster in chicken samples than in pork samples and faster at higher temperatures. Based on these results, it was concluded that, especially in pork samples, there is a risk of false-positive PCR results. This was confirmed in a quantitative study on cell death and signal persistence over a period of 28 days, employing three different methods, i.e. viable counts, direct qPCR, and finally floatation, a recently developed discontinuous density centrifugation method, followed by qPCR. Results showed that direct qPCR resulted in an overestimation of up to 10 times of the amount of cells in the samples compared to viable counts, due to detection of DNA from dead cells. However, after using floatation prior to qPCR, results resembled the viable count data. This indicates that by using of floatation as a sample treatment step prior to qPCR, the risk of false-positive PCR results due to detection of dead cells, can be minimized.
Faster quantitative real-time PCR protocols may lose sensitivity and show increased variability.
Hilscher C, Vahrson W, Dittmer DP.
Nucleic Acids Res. 2005 Nov 27;33(21):e182.
Department of Microbiology and Immunology and Lineberger Comprehensive Cancer
Center, The University of North Carolina at Chapel Hill, NC, USA.
Quantitative real-time PCR has become the method of choice for measuring mRNA transcription. Recently, fast PCR protocols have been developed as a means to increase assay throughput. Yet it is unclear whether more rapid cycling conditions preserve the original assay performance characteristics. We compared 16 primer sets directed against Epstein-Barr virus (EBV) mRNAs using universal and fast PCR cycling conditions. These primers are of clinical relevance, since they can be used to monitor viral oncogene and drug-resistance gene expression in transplant patients and EBV-associated cancers. While none of the primers failed under fast PCR conditions, the fast PCR protocols performed worse than universal cycling conditions. Fast PCR was associated with a loss of sensitivity as well as higher variability, but not with a loss of specificity or with a higher false positive rate.
RT-PCR: considerations for efficient and sensitive assay design
Real-time RT-PCR has been recognised as an accurate and sensitive method of quantifying mRNA transcripts. Absence of post amplification procedures allows rapid analysis with a greater sample throughput, yet with less risk of amplicon carry-over as reaction tubes are not opened. In order to maximise sensitivity, careful reaction design and optimisation is essential. Several aspects of assay design for real-time RT-PCR are discussed in this paper. We demonstrate the effect of amplicon secondary structure on reaction efficiency and its importance for primer design. Taqman probes with a deoxyguanosine base at the 5Vend fluoresce weakly when labelled with FAM, although weak fluorescence is not a problem when probes are labelled with Texas Red. DNA contamination of RNA samples purified using silica membrane columns is a significant problem but DNase digestion can be used to reduce this, particularly in-solution. MMLV and AMV enzyme systems using a variety of RT priming methods are compared and the problem of primer–dimer formation associated with RT enzymes is described.
Gene expression analysis by real-time reverse
transcription polymerase chain reaction: influence of tissue handling
Factors such as warm ischemia and time at room temperature before tissue treatment may inXuence the results of mRNA expression analyses on tissue specimens obtained during surgery. We evaluated the eVect of these factors on RNA integrity and mRNA expression levels by incubating freshly obtained mouse liver tissue at 25 or 37 °C for periods of 0–4 h. Changes in the mRNA expression levels of seven genes, Tbp, Eef1a, Fos, Junb, Myc, Vegf, and Glut2, were determined by real-time reverse transcription-polymerase chain reaction. Incubation at 25 °C for up to 4 h only slightly altered (by a factor of less than 2) levels of mRNA for Tbp, Eef1a, Junb, Myc, Vegf, and Glut2. This result is consistent with limited RNA degradation at this temperature. Incubation at 37 °C strongly aVected the levels of these mRNAs. Four hours of incubation at this temperature resulted in extensive RNA degradation, with mRNA levels falling to 1/10th those before incubation. When relative quantiWcation was performed, i.e., quantiWcation of the target gene transcripts in comparison to an endogenous housekeeping transcript (Tbp or Eef1a), the changes in mRNA levels were reduced to less than 2.5-fold. Fos behaved very diVerently from the other genes tested on incubation, with Fos mRNA levels increasing considerably following incubation at either 25 or 37 °C. Our data suggest that, with the exception of certain genes induced by tissue injury, relative quantiWcation of mRNA, even on degraded RNA samples, can provide a reliable estimate of in vivo mRNA levels.
Development and validation
of an externally standardised quantitative Insulin like
Technical Note No. LC 1/1999
elimination of primer-dimer accumulation in PCR
T.B. Balla F.A. Plummerb K.T. HayGlassa
Int Arch Allergy Immunol 2003;130: 82–86
Clin Chem Lab Med 2000 Feb;38(2):87-91
Switzerland, Molecular Systems, Rotkreuz
In the very beginning of polymerase chain reaction (PCR) tests entering the field of diagnosis of infectious agents, the introduction of this technology into routine diagnosis was hampered by its frequent tendency to create false-positive results because of contamination. This problem is now widely solved by the introduction of the uracil-N-glycosylase (UNG) anticontamination technology. However, care must still be taken to avoid other sources of producing false positive results. They might additionally derive from human error and/or insufficient PCR amplification and detection protocols. A special case lies in the fact that PCR also amplifies DNA from dead organisms rendering a result diagnostically correct as positive, but clinically as false-positive. In PCR, as in any other diagnostic test, the risk of creating a false-negative result also exists. In such a case, the most probable source besides human error, low target or poor amplification and detection protocols is an inhibition caused by interfering substances in a patient's sample. Strategies to recognize and overcome this issue are discussed in this article. Finally a few results from quality control studies on amplification technologies in the diagnosis of infectious agents are reviewed.
Teo IA, Choi JW, Morlese J, Taylor G, Shaunak S.
J Immunol Methods 2002 Dec 1;270(1):119-33
Department of Infectious Diseases, Division of Investigative Science, Faculty of
Medicine, Imperial College at Hammersmith Hospital, Ducane Road, London W12 ONN, UK.
The LightCycler is a rapid air-heated thermal cycler which incorporates a fluorimeter for the detection and quantification of Polymerase Chain Reaction (PCR) amplified products. It provides real-time cycle-by-cycle analysis of product generation. Amplification occurs in glass capillary tubes. The products are detected using a fluorescent double stranded DNA binding dye or fluorescent probes. However, conditions that work well in conventional PCR reactions do not readily translate to the LightCycler. Whilst using this new technology to study an infectious pathogen in human tissue samples, several parameters were identified which can have an adverse effect on the reliable and reproducible quantification of low copy number target DNA. They included abstraction of PCR reagents on glass, primer-dimer formation, non-specific product generation, and a failure to amplify low copy number target when it is present in a high background of human chromosomal DNA. For each problem identified, several solutions are described. Novel approaches are also described to ensure that amplification of target DNA and of the quantification standards occurs with the same efficiency. With appropriate changes to the protocols currently in use, LightCycler quantitative Polymerase Chain Reaction (LC-qPCR) can be used to achieve a level of accuracy that exceeds that of an enzyme immunoassay. The LC-qPCR optimisation strategies described are of particular relevance when applying this technology to the study of pathogens in tissue samples. The technique offers the enormous potential for reliable and reproducible quantitative PCR of low copy number target DNA.
Evaluation of a Homemade SYBR® Green I Reaction Mixture for Real-Time PCR Quantification of Gene Expression.
Albert Karsai, Sabine Müller, Stefan Platz, and Marie-Theres Hauser (2002)
University of Agricultural, Sciences Vienna, Austria
Real-time PCR is an accurate method that can be used for the quantification of specific DNA molecules. Here we provide a protocol for SYBR® Green I in real-time PCR applications using plastic reaction tubes. We report that SYBR Green I is alkali labile and once degraded inhibits the PCR. In our optimized protocol, diluted aliquots of SYBR Green I remain stable for at least two weeks. We also evaluated different cDNA synthesis protocols for the quantification of multiple genes from the same cDNA preparation. The best result was obtained with cDNAs synthesized by OmniScript reverse transcriptase from 2.5 µg total RNA using oligo d(T)18 primers. The cDNA reactions could be diluted 1:25, allowing the quantification of up to 125 different medium expressed genes of Arabidopsis. Extension times ranged between 20 and 40 bp/s for accurate quantification of PCR products up to approximately 400 bp in the Rotor-Gene 2000 system. Using our optimized real-time PCR protocol, the reproducibility and amplification efficiency was high and comparable to a commercially available SYBR Green I kit. Furthermore, the sensitivity allowed us to quantify 10–20 copies of mRNA and dsDNA. Thus, the protocol eliminates the need for expensive pre-made kits.
Nucleic Acids Res 2002 Sep 1;30(17): e89
University Laboratory of Physiology and MRC Anatomical Neuropharmacology Unit,
Department of Pharmacology, Oxford University, Parks Road, Oxford OX1 3PT, UK.
The real-time quantitative polymerase chain reaction (rtqPCR) has overcome the limitations of conventional, time-consuming quantitative PCR strategies and is maturing into a routine tool to quantify gene expression levels, following reverse transcription (RT) of mRNA into complementary DNA (cDNA). Expression profiling with single-cell resolution is highly desirable, in particular for complex tissues like the brain that contain a large variety of different cell types in close proximity. The patch-clamp technique allows selective harvesting of single-cell cytoplasm after recording of cellular activity. However, components of the cDNA reaction, in particular the reverse transcriptase itself, significantly inhibit subsequent rtqPCR amplification. Using undiluted single-cell cDNA reaction mix directly as template for rtqPCR, I observed that the amplification kinetics of rtqPCRs were dramatically altered in a non-systematic fashion. Here, I describe a simple and robust precipitation protocol suitable for purification of single-cell cDNA that completely removes inhibitory RT components without detectable loss of cDNA. This improved single-cell real-time RT-PCR protocol provides a powerful tool to quantify differential gene expression of individual cells and thus could complement global microarray-based expression profiling strategies.
Rapid, single-tube method for quantitative preparation and analysis of RNA andBACKGROUND: Current methods for accurate quantification of nucleic acids typically begin with a template preparation step in which DNA and/or RNA are freed of bound proteins and are then purified. Isolation of RNA is particularly challenging because this molecule is sensitive to elevated temperatures and is degraded by RNases, which therefore have to be immediately inactivated upon cell lysis. Many protocols for nucleic acids purification, reverse transcription of RNA and/or amplification of DNA require repeated transfers from tube to tube and other manipulations during which materials may be lost.
DNA in samples as small as one cell.
Hartshorn C, Anshelevich A, Wangh LJ.
BMC Biotechnol. 2005 Jan 13;5(1):2.
Department of Biology, Brandeis University, Waltham, MA 02454-9110, USA.
RESULTS: This paper introduces a novel and highly reliable single-tube method for rapid cell lysis, followed by quantitative preparation and analysis of both RNA and/or DNA molecules in small samples. In contrast to previous approaches, this procedure allows all steps to be carried out by sequential dilution in a single tube, without chemical extraction or binding to a matrix. We demonstrate the utility of this method by quantification of four genes, Xist, Sry and the two heat-inducible hsp70i (hsp70.1 and hsp70.3), as well as their RNA transcripts in single mouse embryos and in isolated blastomeres.
CONCLUSION: This method virtually eliminates losses of nucleic acids and is sensitive and accurate down to single molecules.
Potential influence of the first PCR cycles in real-time comparative gene quantifications.
There is an underlying assumption in real-time PCR that the amplification efficiency is equal from the first cycles until a signal can be detected. In this study, we evaluated this assumption by analyzing genes with known gene copy number using real-time PCR comparative gene quantifications. Listeria monocytogenes has six 23S rRNA gene copies and one copy of the hlyA gene. We determined 23S rRNA gene copy numbers between 0.9 and 1.6 relative to hlyA when applying the comparative gene quantification approach. This paper focuses on the first cycles of PCR to explain the difference between known and determined gene copy numbers. Both theoretical and experimental evaluations were done. There are three different products (types 1-3) dominating in the first cycles. Type 1 is the original target, type 2 are undefined long products, while type 3 are products that accumulate during PCR. We evaluated the effects of type 1 and 2 products during the first cycles by cutting the target DNA with a restriction enzyme that cuts outside the boundaries of the PCR products. The digestion resulted in a presumed increased amplification efficiency for type 1 and 2 products. Differences in the amplification efficiencies between type 1, 2, and 3 products may explain part of the error in the gene copy number determinations using real-time PCR comparative gene quantifications. Future applications of real-time PCR quantifications should account for the effect of the first few PCR cycles on the conclusions drawn.
Reagent volume and plate bias in real-time polymerase chain reaction.
When optimal conditions are employed, real-time polymerase chain reaction (PCR) is a sensitive and accurate technique enabling the quantiWcation of low-copynumber transcripts. However, as with conventional PCR, small variations in initial reaction conditions are amplified exponentially and can signiWcantly aVect results. Uniform reaction conditions are therefore essential to achieve accuracy and reproducibility during transcript quantiWcation. Reagent costs are considerable, and in a high-throughput setting a reduction in the reagent volume used in each reaction would signiWcantly reduce the cost of real-time PCR. However, the eVect of reduced volume on the accuracy of results in a plate-based system has not, to our knowledge, been examined. Here we show that lower reagent volumes can reduce reproducibility by enhancing a bias in results across a plate.
geometric averaging of multiple internal control genes.
Vandesompele J., De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F (2002)
Genome Biology 2002; 3(7): 0034.I - 0034.II
Background: Gene-expression analysis is increasingly important in biological research, with real-time reverse transcriptionPCR (RT-PCR) becoming the method of choice for high-throughput and accurate expression profiling of selected genes. Given the increased sensitivity, reproducibility and large dynamic range of this methodology, the requirements for a proper internal control gene for normalization have become increasingly stringent. Although housekeeping gene expression has been reported to vary considerably, no systematic survey has properly determined the errors related to the common practice of using only one control gene, nor presented an adequate way of working around this problem.
Results: We outline a robust and innovative strategy to identify the most stably expressed control genes in a given set of tissues, and to determine the minimum number of genes required to calculate a reliable normalization factor. We have evaluated ten housekeeping genes from different abundance and functional classes in various human tissues, and demonstrated that the conventional use of a single gene for normalization leads to relatively large errors in a significant proportion of samples tested. The geometric mean of multiple carefully selected housekeeping genes was validated as an accurate normalization factor by analyzing publicly available microarray data.
Conclusions:The normalization strategy presented here is a prerequisite for accurate RT-PCR expression profiling, which, among other things, opens up the possibility of studying the biological relevance of small expression differences.
using SYBR green I depends on cDNA synthesis conditions.
Lekanne Deprez RH, Fijnvandraat AC, Ruijter JM, Moorman AF. (2002)
Anal Biochem. 2002 307(1): 63-69.
development of real-time PCR has offered the opportunity of sensitive
quantification of mRNA levels that is crucial in biomedical
reverse transcription (RT)-PCR is at present the most sensitive method available,
many low abundant mRNAs are, although detectable, often not quantifiable. Here we
report an improved two-step real-time RT-PCR procedure using SYBR green I
the LightCycler that better permits accurate quantification of mRNAs.
Omission of dithiothreitol from the cDNA synthesis reaction was found to
be crucial. This resulted in a lower cycle number at which
the cDNA level is
determined (C(T) value), steeper amplification curves, and
background fluorescence in the subsequent PCR. In addition, the
choice of the cDNA
priming oligo can improve detection sensitivity even further.
In contrast to
hexamer primer usage, both gene-specific and oligo-dT(VN) priming
were very efficient
and accurate, with gene-specific priming being the most sensitive. Finally,
accurate quantification of mRNAs by real-time PCR using
I requires verification of the specificity of PCR by both melting curve
and gel analysis.