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Overview: Real-time PCR, also known as kinetic PCR, qPCR, qRT-PCR and RT-qPCR, is quantitative PCR method for the determination of cope number of PCR templates such as DNA or cDNA in a PCR reaction. There are two flavors of real-time PCR: probe-based and intercalator-based. Both methods require a special thermocycler equipped with a sensitive camera that monitors the fluorescence in each well of the 96-well plate at frequent intervals during the PCR Reaction. Probe-based real-time PCR, also known as TaqMan PCR, requires a pair of PCR primers as regular PCR does, an additional fluorogenic probe which is an oligonucleotide with both a reporter fluorescent dye and a quencher dye attached. Intercalator-based method, also known as SYBR Green method, requires a double-stranded DNA dye in the PCR reaction which binds to newly synthesized double-stranded DNA and gives fluorescence. TaqMan method is more accurate and reliable than SYBR green method, but also more expensive.
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Protocols
  • Protocol for Real-Time RT-PCR (Dr. Xiaowei Wang at the Department of Molecular Biology, Massachusetts General Hospital)
    This protocol describes the detailed experimental procedure for real-time RT-PCR using SYBR Green I. The procedure begins with reverse transcription of total RNA. The cDNA is then used as template for real-time PCR with gene specific primers. You may need to modify this protocol if you use different reagents or instruments for real-time PCR.
    http://pga.mgh.harvard.edu/primerbank/PCR_protocol...
    Added: Fri May 14 2004, Hits: 36616, Reviews: 0  Write review   Cached
  • Real-Time PCR (M.Tevfik Dorak, MD, PhD)
    Excellent guide to real-time PCR including background; TaqMan primer and probe design guidelines; General recommendations for real-time RT-PCR;Setting up one-step TaqMan reaction;Cycling parameters for one-step PCR;Interpretation of results; The comparative CT method (DDCT) for relative quantitation of gene expression; Points to remember and trouble shooting; and more... (alternative url: http://home.att.net/~dorak/genetics/realtime.html)
    http://dorakmt.tripod.com/genetics/realtime.html
    Added: Sun Sep 22 2002, Hits: 51331, Reviews: 0  Write review   Cached
  • Real-Time or Kinetic PCR (University of Iowa DNA Facility)
    This page describes the following topics on real-time PCR:
    • Real-Time Chemistry
    • Instrumentation
    • Real-Time PCR Quantitation
    • Primer and Probe Design 
    • Thermal Cycling Parameters 
    • Sample Preparation
    • Data and Analysis
    • Supplemental Information

    http://dna-9.int-med.uiowa.edu/realtime.htm
    Added: Sun Sep 22 2002, Hits: 21740, Reviews: 0  Write review   Cached
  • Theory, principles and background info Real-Time PCR (qPCR) Basics (Rob Powell, Brooke Laboratory, RCMB, Southampton General Hospital)
    A good place for you to learn real-time PCR. Topics included are as follows:
    Introduction to 'Real time' PCR
    Assay parameters
       Melt Curves and Primer Dimers
       Standard Curves
       Dynamic Range
    Quantification methods
      Introduction
      Delta CT
      Standard Curve

    http://www.primerdesign.co.uk/primerdesign/qPCR_ba...
    Added: Sun Jul 10 2005, Hits: 9048, Reviews: 0  Write review   Cached
  • FAQs on Real-Time RT-PCR (Dr. Xiaowei Wang at the Department of Molecular Biology, Massachusetts General Hospital)
    Some questions and answers on real-time PCR primer design and amplification
    http://pga.mgh.harvard.edu/primerbank/faq.html
    Added: Fri May 14 2004, Hits: 11766, Reviews: 0  Write review   Cached
  • overviews on quantitative kinetic RT-PCR PDF file (Dr. Michael W. Pfaffl, Gene Quantification )
    This review discusses the technical aspects involved, contrasts conventional and kinetic RT-PCR methods for quantitating gene expression and compares the different kinetic RT-PCR systems. It illustrates the usefulness of these assays by demonstrating the significantly different levels of transcription between individuals of the housekeeping gene family, glyceraldehyde-3-phosphate-dehydrogenase (GAPDH).
    http://www.gene-quantification.de/bustin-2000.pdf
    Added: Sun Sep 22 2002, Hits: 11682, Reviews: 1 Read reviews  Write review  
  • Quantitative Real-Time RT-PCR (Laboratory of Dr. Bart Frank, Oklahoma Medical Research Foundation)
    cDNA synthesis and real-time PCR amplification using the SYBRŪGreen I dye assay chemistry
    http://omrf.ouhsc.edu/~frank/M_QPCR-OMNISCRIPT-RT....
    Added: Mon Dec 19 2005, Hits: 7795, Reviews: 0  Write review   Cached
  • Theory, principles and background info Real-time PCR in Virology PDF file (Dr. Michael W. Pfaffl, Gene Quantification )
    This paper describes the background, advantages and limitations of real-time PCR
    http://www.gene-quantification.de/mackay-2002.pdf
    Added: Sun Sep 22 2002, Hits: 7236, Reviews: 0  Write review  
  • Real-Time PCR Protocols (Rob Powell, Brooke Laboratory, RCMB, Southampton General Hospital)
    Mutiple protocols from RNA isolation, DNase treatment, RT reaction and real-time PCR reaction.
    http://www.primerdesign.co.uk/primerdesign/mol_bio...
    Added: Sun Jul 10 2005, Hits: 3997, Reviews: 0  Write review   Cached
  • Real-Time PCR Quantification Using Cloned Standards and Multiple Housekeeping Genes (Protocol Online)
    We reported a general laboratory procedure for QRT-PCR, which addresses most of the inherent difficulties with this technique, can be easily adopted by any laboratory.
    Added: Tue Jun 26 2007, Reviews: 0  Write review  
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