real-time PCR
optimisation (4) 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. Longevity
Methods
to Control ContaminationB. Detection C. Amplification Detection of Contamination A. Reagent-Only
Controls
Concepts
of Contamination ControlA. 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
Dimers
General
Contamination Control Procedures2. Pyrimidine: Psoralen Photoadducts by UV A. Laboratory
Construction
B. Environmental Control C. Laboratory Equipment and Personnel D. Flow of Samples Summary
References 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 [1]. 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 [2]. 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.
|