Technique / Molecular Biology / PCR / Quantitative real-time PCR

Real-time PCR is a quantitative qPCR used to amplify and quantify DNA molecules in given samples. Real-time RT-PCR is the commonly used technique to quantify mRNA expression.

Protocols for real time PCR titration new protocol
A titration protocol for real-time PCR with figures (Dr Patrick SALMON, Geneva School of Medicine). ...

Genotyping mice by real-time qPCR new protocol
Real-time PCR prototocol for genotyping Ts65Dn mice (Don Liu, Cecilia Schmidt, Tim Billings, and Muriel T. Davisson, The Jackson Laboratory). Genotyping Ts65Dn mice is based on doing simultaneous quantitative PCR amplification of a gene or genes in the Ts65 ...

Patti Lab Taqman Real time PCR protocol new protocol
Real-time quantitative multiplex RT -PCR (Taqman) protocol. This is performed as a 2-step reaction: 1. cDNA synthesis from DNase I treated total RNA 2. multiplex real-time taqman PCR optimization and PCR. This is a very detailed Taqman real time RT-PCR ...

Real-time PCR data analysis and reviews new recommended review
A technical overview on real-time PCR including introduction, primer probe design, data analysis and some recent pdf review articles. (DNA facility, University of Iowa) ...

Absolute quantification in real-time RT-PCR new recommended review
A nice review and collection of technique articles on absolute real-time PCR quantification. (edited by Michael W. Pfaffl) ...

Real-time PCR introduction new review
A introduction of real-time PCR by M.Tevfik Dorak, MD, PhD. It gives background information on real-time PCR, guidlines on designing probe/primer and conditions on performing PCR. ...

Real-Time PCR Method review
A background introduction on real-time PCR technique (Davidson college, Dept. of Biology). Real-time PCR is able to detect sequence-specific PCR products as they accumulate in "real-time" during the PCR amplification process. As the PCR product of interest ...

Real-Time PCR Vs. Traditional PCR review
This PDF tutorial will discuss the evolution of traditional PCR methods towards the use of Real-Time chemistry and instrumentation for accurate quantitation (Applied biosystems). ...

SYBR Green Real-time PCR new review
A pdf powerpoint overview on SYBR green real-time PCR. It discussed different methods, data analysis using delta-CT etc (47 pages). (www.gene-quantification.info) SYBR-green real-time is perhaps the best way to quantify gene expression in large scale with h ...

Introduction to Quantitative PCR: Methods and Application Guide new review
Stratagen real-time PCR techbook. It is downloadable as a 50 pages pdf file. Table of Contents: Real-Time vs. Endpoint Quantitative PCR ............................................................................................................... ...

Ten Most Common Real-Time qRT-PCR Pitfalls new review
List of 10 possible reasons for an unsuccessful real-time PCR (Ambion inc.) More detailed description can be found from this page. Poor primer and probe design. Using poor quality RNA. Not using "master mixes". Introducing cross-contamination. Not u ...

Real time PCR troubleshooting
We are planning to have a new Real Time PCR machine in our lab but still in puzzle to find the best. Can anyone ... ... ...

Semi-quantitative PCR troubleshooting
I have to do semi-quantitative PCR in order to study the regulation of expression of receptors mRNA. I've already done optimization of annealing temp., MgCl2 conc., etc... If I want to compare the level of two different mRNA in response of a stimul ...

Real-time PCR protocol and troubleshooting troubleshooting
Real-time PCR protocol with troubleshooting guide (from primersbank) Reference: Xiaowei Wang and Brian Seed (2003) A PCR primer bank for quantitative gene expression analysis. Nucleic Acids Research 31(24): e154; pp.1-8. ...

Real Time PCR Primer and Probe Database new recommended database
This is a public database holding real time PCR primers and probes for popular chemistries (SYBR Green I, Taqman, Hybridisation Probes, Molecular Beacon) to prevent time-consuming primer design and experimental optimisation. Reference: PATTYN, F., SPELE ...

PCR Primer bank For Gene Expression Detection or Quantification database
PrimerBank is a public resource for PCR primers. These primers are designed for gene expression detection or quantification (real-time PCR). PrimerBank contains about 180,000 primers covering most known human and mouse genes. There are several ways to search f ...

Real-time qPCR Interactive Tutorial new media
Biocompare's qPCR Tutorial presents researchers with an overview of real-time qPCR, identifies the advantages and disadvantages of the various detection technologies, outlines the key issues for optimizing experimental design and offers a brief description of ...

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High-throughput real-time quantitative reverse transcription PCR.
(Curr Protoc Mol Biol. 2006 Feb;Chapter 15:Unit 15.8.)
Bookout AL, Cummins CL, Mangelsdorf DJ, Pesola JM, Kramer MF.
Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas, USA.

Extensive detail on the application of the real-time quantitative polymerase chain reaction (QPCR) for the analysis of gene expression is provided in this unit. The protocols are designed for high-throughput, 384-well-format instruments, such as the Applied Biosystems 7900HT, but may be modified to suit any real-time PCR instrument. QPCR primer and probe design and validation are discussed, and three relative quantitation methods are described: the standard curve method, the efficiency-corrected DeltaCt method, and the comparative cycle time, or DeltaDeltaCt method. In addition, a method is provided for absolute quantification of RNA in unknown samples. RNA standards are subjected to RT-PCR in the same manner as the experimental samples, thus accounting for the reaction efficiencies of both procedures. This protocol describes the production and quantitation of synthetic RNA molecules for real-time and non-real-time RT-PCR applications.

Application of Real-time Polymerase Chain Reaction RT-PCR (Technique review)
(Hongbao Ma et.al., Michigan State University)
Abstract: The real-time polymerase chain reaction (RT-PCR), also called quantitative real-time polymerase chain reaction (QRT-PCR) or kinetic polymerase chain reaction (kPCR), is a technique used to simultaneously quantify and amplify a DNA molecule. It is used to determine whether a specific DNA sequence is present in the sample; and if it is present, the number of copies in the sample. It is the real-time version of quantitative polymerase chain reaction (qPCR), itself a modification of polymerase chain reaction (PCR). The procedure of RT-PCR follows the regular PCR procedure, but the DNA is quantified after each round of amplification. Two common methods of quantification are the use of fluorescent dyes that intercalate with double-strand DNA, and modified DNA oligonucleotide probes that fluoresce when hybridized with a complementary DNA. RT-PCR could be combined with reverse transcription polymerase chain reaction to quantify messenger RNA (mRNA) at a particular time for in a particular cell or tissue type. [The Journal of American Science. 2006;2(3):1-15].

(Quantitative real-time PCR reviews)
1: Libus J, Storchova H.
Quantification of cDNA generated by reverse transcription of total RNA provides
a simple alternative tool for quantitative RT-PCR normalization.
Biotechniques. 2006 Aug;41(2):156, 158, 160 passim. Review. No abstract
available.

2: Stirling D.
Qualitative and quantitative PCR: a technical overview.
Methods Mol Biol. 2003;226:181-4. Review.

3: Joyce C.
Quantitative RT-PCR. A review of current methodologies.
Methods Mol Biol. 2002;193:83-92. Review.

4: Salonga DS, Danenberg KD, Grem J, Park JM, Danenberg PV.
Relative gene expression in normal and tumor tissue by quantitative RT-PCR.
Methods Mol Biol. 2002;191:83-98. Review.

5: Jung R, Soondrum K, Neumaier M.
Quantitative PCR.
Clin Chem Lab Med. 2000 Sep;38(9):833-6. Review.

6: Freeman WM, Walker SJ, Vrana KE.
Quantitative RT-PCR: pitfalls and potential.
Biotechniques. 1999 Jan;26(1):112-22, 124-5. Review.

7: Orlando C, Pinzani P, Pazzagli M.
Developments in quantitative PCR.
Clin Chem Lab Med. 1998 May;36(5):255-69. Review.

8: Lie YS, Petropoulos CJ.
Advances in quantitative PCR technology: 5' nuclease assays.
Curr Opin Biotechnol. 1998 Feb;9(1):43-8. Review.

9: Zimmermann K, Mannhalter JW.
Technical aspects of quantitative competitive PCR.
Biotechniques. 1996 Aug;21(2):268-72, 274-9. Review.

10: Reischl U, Kochanowski B.
Quantitative PCR. A survey of the present technology.
Mol Biotechnol. 1995 Feb;3(1):55-71. Review.

11: Clementi M, Menzo S, Bagnarelli P, Manzin A, Valenza A, Varaldo PE.
Quantitative PCR and RT-PCR in virology.
PCR Methods Appl. 1993 Feb;2(3):191-6. Review.

12: Provenzano M, Mocellin S.
Complementary techniques: validation of gene expression data by quantitative
real time PCR.
Adv Exp Med Biol. 2007;593:66-73. Review.

13: Utermark J, Karlovsky P.
Quantification of green fluorescent protein fluorescence using real-time PCR
thermal cycler.
Biotechniques. 2006 Aug;41(2):150, 152, 154. Review.

14: Pinzani P, Orlando C, Pazzagli M.
Laser-assisted microdissection for real-time PCR sample preparation.
Mol Aspects Med. 2006 Apr-Jun;27(2-3):140-59. Epub 2006 Feb 9. Review.

15: Gibson NJ.
The use of real-time PCR methods in DNA sequence variation analysis.
Clin Chim Acta. 2006 Jan;363(1-2):32-47. Epub 2005 Sep 22. Review.

16: Valasek MA, Repa JJ.
The power of real-time PCR.
Adv Physiol Educ. 2005 Sep;29(3):151-9. Review.

17: Wong ML, Medrano JF.
Real-time PCR for mRNA quantitation.
Biotechniques. 2005 Jul;39(1):75-85. Review.

18: Rooney PH.
Multiplex quantitative real-time PCR of laser microdissected tissue.
Methods Mol Biol. 2005;293:27-37. Review.

19: Mocellin S, Rossi CR, Marincola FM.
Quantitative real-time PCR in cancer research.
Arch Immunol Ther Exp (Warsz). 2003;51(5):301-13. Review.

20: Ong YL, Irvine A.
Quantitative real-time PCR: a critique of method and practical considerations.
Hematology. 2002 Feb;7(1):59-67. Review.

21: Klein D.
Quantification using real-time PCR technology: applications and limitations.
Trends Mol Med. 2002 Jun;8(6):257-60. Review.

22: Lehmann U, Kreipe H.
Real-time PCR analysis of DNA and RNA extracted from formalin-fixed and
paraffin-embedded biopsies.
Methods. 2001 Dec;25(4):409-18. Review.

Averages fold changes in real-time PCR
(Forum)
Questions on fold changes for the real-time PCR statistical analysis

problems with housekeepings in real-time pcr
(Forum)
house-keeping gene for real-time PCR internal control

Signal Pattern Problem in Real-time PCR
(Forum)
Real-time PCR signal problem with molecular beacon probes