New papers for optimising your qPCR:
Standardization of real-time PCR gene expression data from independent biological replicates
Erik Willems ab*, Luc Leyns b, Jo Vandesompelec*
a Del E. Webb Neuroscience, Aging, and Stem Cell Research Center, Burnham Institute for Medical Research, La Jolla, CA, USA
b Laboratory for Cell Genetics, Vrije Universiteit Brussel, B-1050 Brussels, Belgium
c Center for Medical Genetics, Ghent University Hospital, B-9000 Ghent, Belgium
Notes & Tips / Anal. Biochem. 379 (2008) 127–129
Gene expression analysis by quantitative reverse transcription PCR (qRT–PCR) allows accurate quantiﬁcations of messenger RNA (mRNA) levels over different samples. Corrective methods for different steps in the qRT–PCR reaction have been reported; however, statistical analysis and presentation of substantially variable biological repeats present problems and are often not meaningful, for example, in a biological system such as mouse embryonic stem cell differentiation. Based on a series of sequential corrections,
including log transformation, mean centering, and autoscaling, we describe a robust and powerful standardization method that can be used on highly variable data sets to draw statistically reliable conclusions.
Control of Contamination Associated with PCR and Other Amplification Reactions.
by Theodore E. Mifflin, Ph.D., DABCC
INTRODUCTION TO CONTAMINATION CONTROL
A molecular diagnostic laboratory that plans on using one or more in-vitro amplification reaction(s) (IVAR) should also be evaluating measures to control a contamination problem which parallels the use of these procedures. Historically, the concept of contamination in a biomedical laboratory has been associated with the unintentional disbursement of radioisotopes (e.g., 32P, 35S, etc.) in areas not designated for their use. Removal of these radioactive species was typically straightforward, rapid, and effective. Indeed, trace amounts of these radioactive species have usually been removed without problem and the laboratory restored to its original non-contaminated condition..........................
TABLE OF CONTENTS
Introduction to Contamination Control
A. LongevityMethods to Control Contamination
Detection of Contamination
A. Reagent-Only ControlsConcepts of Contamination Control
A. Pre-Amplification Contamination Control
1. Positive Displacement Pipettors
2. ART Self-Sealing Barrier Tips
3. Uracil-N-Glycosylase (UNG)
4. 3' Ribonucleotide Amplimer
B. Post Amplification Containment Control
1. UV-induced Thymine DimersGeneral Contamination Control Procedures
2. Pyrimidine: Psoralen Photoadducts by UV
A. Laboratory Construction
B. Environmental Control
C. Laboratory Equipment and Personnel
D. Flow of Samples
Enhancing the efficiency of a PCR using gold nanoparticles.
Min Li, Yu-Cheng Lin*, Chao-Chin Wu and Hsiao-Sheng Liu1
Department of Engineering Science and 1Department of Microbiology and Immunology, National Cheng Kung University, Tainan, Taiwan, Republic of China
Nucleic Acids Research, 2005, Vol. 33, No. 21 e184
We found that the PCR could be dramatically enhanced by Au nanoparticles. With the addition of 10 0.7nMof13nmAunanoparticles into thePCRreagent, the PCR efficiency was increased. Especially when maintaining the same or higher amplification yields, the reaction time could be shortened, and the heating/cooling rates could be increased. The excellent heat 15 transfer property of the nanoparticles should be the major factor in improving thePCRefficiency. Different PCR systems, DNApolymerases,DNAsizes and complexsamples werecompared in this study. Our results demonstrated that Au nanoparticles increase the 20 sensitivity of PCR detection 5- to 10-fold in a slower PCR system (i.e. conventional PCR) and at least 104-fold in a quicker PCR system(i.e. real-time PCR). After the PCR time was shortened by half, the 100 copies/ml DNA were detectable in real-time PCR with gold col-25 loidadded,however, atleast106copies/mlofDNAwere needed to reach a detectable signal level using the PCR reagent without gold colloid. This innovation could improve the PCR efficiency using nonexpensive polymerases, and general PCR reagent. It 30 is a new viewpoint in PCR, that nanoparticles can be used to enhance PCR efficiency and shorten reaction times.
Increasing Detection of Polymerase Chain Reaction (PCR) by Isolation of PCR Products (IPCRp).
Pero Dimsoski, Sam L. Woo
Applied Biosystems, Foster City, Calif, USA
Aim To develop a method for enhanced polymerase chain reaction (PCR) product detection. Methods During the PCR, the double-stranded product is generated with fluorescent dye on one strand, and biotin on the other strand. The product is captured on the streptavidin-coated plates with high efficiency (IPCRp).Washing of the all unamplified compounds, including dye-labeled unincorporated primers, follows the PCR. The targeted dye-labeled PCR product is released by denaturation and loaded on the detection platform. Results After the application of the IPCRp, the resulting product is highly concentrated targeted dye-labeled single-strandedDNA,free of the unincorporated primers and other PCRartifacts. The strength of the signal of the IPCRp product on detection platform is two- to five-fold higher than the strength of the signal of the conventional PCR product. Conclusion The IPCRp procedure can be accomplished in less than 20minutes. Efficient isolation of the PCR products has two steps, washing and denaturation. It can increase the yield of targeted PCR product and increase the sensitivity of the detection platform.
Increased efficiency of genetic profiling through quantity and quality assessment of fluorescently labeled oligonucleotide primers.
Timothy R. Frasier and Bradley N. White
Trent University, Peterborough, ONT, Canada
BioTechniques 44:49-52 (January 2008)
Optimizing the amount of primer to use in PCR amplification is one of the most impor-tant steps when developing protocols for genetic profiling, where subtle changes in primer concentration result in major impacts on the amount of desired product that is amplified. However, there are frequently discrepancies between the reported and actual quantity of primers delivered by suppliers, resulting in a need for re-optimization of conditions between primer orders and limiting the ability to standardize conditions between laboratories. To increase the consistency of genetic profiling protocols, we have developed a simple method to assess the quantity and quality of fluorescently labeled primers and therefore standardize reaction conditions through time and across laboratories. The method is based on analysis by electrophoresis with an automated fluorescent DNA analyzer.
Primers with 5′ flaps improve real-time PCR.
Irina Afonina, Irina Ankoudinova, Alan Mills, Sergey Lokhov, Phan Huynh, and Walt Mahoney
Nanogen, Bothell, WA, USA
BioTechniques 43:770-774 (December 2007)
Primers that contain portions noncomplementary to the target region are usually used to add to the PCR product a utility sequence such as a restriction site or a universal probe binding site. We have demonstrated that primers with short 5′ AT-rich overhangs increase real-time PCR fluorescent signal. The improvement is particularly significant for difficult to amplify templates, such as highly variable viral sequences or bisulfite-treated DNA.
A real-time polymerase chain reaction-based evaluation of cDNA synthesis priming methods.
David Resuehr* and Andrej-Nikolai Spiess
Institute for Hormone and Fertility Research, Centre of Innovative Medicine, Falkenried 88, 20251 Hamburg, Germany
Notes & Tips Analytical Biochemistry 322 (2003) 287–291
Before generating cDNA in a molecular biology lab, the researcher is confronted with choosing the appropriate priming method for the previously isolated RNA. The most common methods utilize the poly(A) tail of the mRNA either by priming with an oligo(dT) primer or by using random hexamers that statistically bind to the mRNA pool. Another method uses gene-speciﬁc primers, but this shall not be discussed here, as the resulting cDNA is limited to one transcript. To obtain a good representation of sequences at the 30 end of the transcripts, it is desirable for the priming to be as far toward the 30 end as possible, for instance by using an oligo(dT) primer to the poly(A) tail of eukaryotic mRNA. On the other hand it is a known and common problem for this method to get a good representation of transcript regions that are upstream at the 50 end of the transcripts . Secondary structures of mRNA can often
cause diﬃculties for the reverse transcriptase, causing the enzyme to stall and end its synthesis well ahead of the 50 end . The beneﬁt of random hexamers is a cDNA synthesis occurring along the entire length of the transcripts, avoiding possible secondary structures such as loops and stems and resulting in a more even representation of the whole mRNA sequence. Here we describe the eﬀect of these two diﬀerent cDNA priming methods on the transcript variety and length, when used individually or in combination, by
evaluating the cDNA synthesis eﬃciency using a quantitative real-time PCR approach.
Comparison of quantitative competitive polymerase chain reaction–enzyme-linked immunosorbent assay with LightCycler-based polymerase chain reaction for measuring cytomegalovirus DNA in patients after hematopoietic stem cell transplantation.
Marketa Pumannovaa, Katerina Roubalova aT, Antonin Vitek b, Jana Sajdova b
a National Institute of Public Health, 100 42, Prague, Czech Republic
b Institute of Haematology and Blood Transfusion, 128 20, Prague, Czech Republic
Diagnostic Microbiology and Infectious Disease (2005)
Development of highly sensitive quantitative assays for cytomegalovirus (CMV) DNA detection is crucial for identification of
immunodeficient patients at high risk of CMV disease. We designed 2 internally controlled competitive quantitative assays, enzyme-linked immunosorbent assay (ELISA)-based and real-time polymerase chain reaction (PCR) tests, using amplification of the same segment of the CMV genome. The aim of this study was to compare sensitivity, specificity, and laboratory performance characteristics of these assays. In both assays, a 159-bp segment of UL83 gene was amplified. External and internal controls were constructed by cloning the amplification product and heterogenous DNA segment flanked by target sequences for CMV-derived primers into bacterial plasmids, respectively. Realtime PCR was performed on LightCycler (Roche Diagnostics, Mannheim, Germany), and amplicons were detected using fluorescence resonance energy transfer probes. Alternatively, PCR products were labeled by digoxigenin, hybridized to immobilized probes, and detected by ELISA. The assays were tested on genomic DNA isolated from laboratory strains of CMV, QCMD control panel, and CMV DNA-positive peripheral blood DNA samples from hematopoietic stem cell transplant recipients, previously characterized by pp65 antigenemia and qualitative nested PCR. Real-time and ELISA-based PCR assays showed a linear course of 1–108 and 10–105 copies of CMV DNA per reaction, respectively. When compared with ELISA-based PCR, real-time PCR showed superiority in inter- and intra-assay reproducibility. Both assays were highly specific in detecting CMV DNA. No difference in amplification efficiency of internal or external standards and wildtype
CMV DNA was found. The assays exhibited 83% concordance in CMV DNA detection from clinical samples, all discrepant samples
having low CMV DNA copy numbers. There was a good correlation between viral DNA loads measured by the 2 assays. Statistically significant correlation was observed between the numbers of CMV DNA copies and pp65-positive leukocytes in the samples tested. Both variants of competitive PCR are adequately sensitive to be used for CMV DNA quantitation in clinical samples. LightCycler PCR, having superior performance characteristics and being less time-consuming, seems to be more suitable for routine diagnosis.
Performance evaluation of thermal cyclers for PCR in a rapid cycling condition.
Young Ho Kim,1 Inchul Yang1, Young-Seuk Bae2, Sang-Ryoul Park1
1Health Metrology Group, Korea Research Institute of Standards and Science,
Daejeon and 2Department of Biochemistry, Kyungpook National University, Daegu, Republic of Korea
BioTechniques 44:495-505 (April 2008)
The performance of thermal cyclers for polymerase chain reactions (PCR) is of great concern in terms of the reliability of PCR-based assays, particularly when rapid cycling conditions are applied to small volume reactions. In this work, the precision of the temperature controls during rapid thermal cycling was measured in 19 commercial thermal cyclers of 8 different models. The temperatures of test solutions in specific locations in each thermal block were simultaneously monitored at 1 s intervals during thermal cycling. A temperature-sensitive multiplex PCR was run in parallel to assess undesirable PCR results caused by poor temperature control. Under the given conditions (20 s of annealing time and 20 μL L reaction volume), a majority of the tested instruments showed prominent curving, undershooting, and/or overshooting in their temperature profiles, which substantially influenced the results of the temperature-sensitive multiplex PCR. Variations between wells were also observed in most instruments. It is strongly hoped that these problems will be addressed by manufacturers and that they will make substantial improvements in the precision and efficiency of thermal cyclers. In the meantime, users of thermal cyclers might be able to avoidunexpected poor outcomes of sensitive PCR-based assays by designing their PCR protocols with these findings in mind.
Sensitivity comparison of real-time PCR probe designs on a model DNA plasmid.
L. Wang a,¤, J.R. Blasic Jr. a, M.J. Holden a, R. Pires b
a Biotechnology Division, National Institute of Standards and Technology, Gaithersburg, MD 20899, USA
b Invitrogen Corporation, Frederick, MD 21704, USA
Analytical Biochemistry 344 (2005) 257–265
<>We investigated three probe design strategies used in quantitative polymerase chain reaction (PCR) for sensitivity in detection of the PCR amplicon. A plasmid with a 120-bp insert served as the DNA template. The probes were TaqMan, conventional molecular beacon (MB), and shared-stem molecular beacon (ATssMB and GCssMB). A shared-stem beacon probe combines the properties of a TaqMan probe and a conventional molecular beacon. It was found that the overall sensitivities for the four PCR probes are in the
order of MB >ATssMB > GCssMB> TaqMan. The Xuorescence quantum yield measurements indicate that incomplete or partialenzymatic cleavage catalyzed by Taq polymerase is the likely cause of the low sensitivities of two shared-stem beacons when compared with the conventional beacon probe. A high-Xuorescence background associated with the current TaqMan probe sequence contributes to the relatively low detection sensitivity and signal-to-background ratio. The study points out that the nucleotide environment surrounding the reporting Xuorophore can strongly aVect the probe performance in real-time PCR.
Real-time RT-PCR and SYBR Green I melting curve analysis for the identification of Plum pox virus strains C, EA, and W: Effect of amplicon size, melt rate, and dye translocation.
Aniko Varga, Delano James
Sidney Laboratory, Canadian Food Inspection Agency, 8801 East Saanich Road, Sidney, BC, Canada V8L 1H3
Journal of Virological Methods 132 (2006) 146–153
Real-time RT-PCR and SYBR green I melt curve analysis of a 74 bp amplicon enabled identification of Plum pox virus strains C, EA, and W, with distinct Tm’s associated with each strain. This test is a useful supplement to a real-time RT-PCR test described earlier that was used to distinguish PPV strains D and M. A longer fragment of 155 bp was not effective for strain identification. A simplified one-tube protocol, with dithiothreitol eliminated from the reaction, showed similar sensitivity when compared to a two-tube protocol. For melt curve analysis, a slower melt rate of 0.1 ◦C/s, compared to 0.4 ◦C/s, was effective for detecting weak amplicons, and improved resolution of the Tm of amplicons amplified simultaneously. SYBR green I was useful for duplex melt curve analysis. In repeated melt run treatments (total of 14) of a single sample containing co-amplified targets, complete translocation of SYBR green I was observed, going from a 74 bp fragment to a 114 bp fragment. The duration of the melt run may be a critical factor affecting SYBR green I binding and translocation, and its manipulation may facilitate improved resolution and simultaneous detection of multiple targets. This phenomenon may explain inconsistent SYBR green I fluorescence patterns associated with melt curve analysis of some amplicon complexes.
Comparison of two standardisation methods in real-time quantitative RT-PCR to follow Staphylococcus aureus genes expression during in vitro growth.
Heidy Eleaume, Said Jabbouri*
Laboratoire de Recherches sur les Biomate´riaux et Biotechnologies, Universite´ du Littoral-Coˆ te d’Opale, Inserm-ERI002,
LR2B, BP 120, F-62327 Boulogne-sur-mer cedex, France
Journal of Microbiological Methods 59 (2004) 363– 370
By real-time quantitative PCR (RTQ-PCR), two different standardisation methods were used to quantify expression of three target genes (RNAII and RNAIII transcripts of agr locus and ica transcript of icaADBC locus): (i) a relative quantification, using a transcript of three housekeeping genes (gyrase A, gyrA; guanylate kinase, gmk and 16S rRNA, 16S) as internal standard, and (ii) an absolute quantification, using cloned sequences of the target genes in known concentrations as external standards. To determine the efficiency and reliability of these two methods, the gene expressions were studied during the growth of a clinical isolate of Staphylococcus aureus. Between 3 and 20 h after inoculation, target gene transcription was analysed using LightCycler Apparatus, LC Data Analysis software and RelQuant software for relative quantification (Roche).For all target genes, the expression profiles obtained with gyrA or gmk as internal standards remained almost identical.However, these profiles varied between each other depending on the standard gene. Due to their important expression variationsduring growth phases, these two housekeeping genes seem inappropriate to be used as internal standards. The absolutequantification of the three transcripts of interest gave results similar to their relative quantification expressed versus 16S rRNA.Therefore, our study suggests the suitable use of 16S rRNA as internal standard in RTQ-PCR quantification of staphylococcal gene expression during the stationary phase of growth.