Top : Molecular Biology : PCR : Real-Time PCR

Summary: 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.
Subcategories
Related Categories:


Protocols
  • Theory, principles and background info Real-Time PCR (qPCR) Basics PDF file (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/Download%20material/...
    Added: Sat Jan 31 2009, Hits: 58209, Reviews: 0  Write review  
  • 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: 40006, Reviews: 0  Write review   Cached
  • SYBR Green Quantitative PCR Protocol (Lusis Lab, UCLA)
    Quantitative PCR is a method used to detect relative or absolute gene expression level. All qPCR involves the use of fluorescence to detect the threshold cycle (Ct) during PCR when the level of fluorescence gives signal over the background and is in the linear portion of the amplified curve. This Ct value is responsible for the accurate quantization of qPCR.
    http://www.genetics.ucla.edu/labs/lusis/greenquant...
    Added: Sat Mar 21 2009, Hits: 32609, Reviews: 0  Write review   Cached
  • Theory, principles and background info Essentials of Real-Time PCR (Life Technologies)
    Things you must know before you do qPCR including basics about Real-Time PCR Assays and Sequence Detection Chemistries SYBRŪ Green I Dye Chemistry About Quantitation Assays Absolute vs. Relative Quantitation Standard Curve Method for Relative Quantitation Comparative CT method for Relative Quantitation Standard Curve Method for Absolute Quantitation
    http://www.invitrogen.com/site/us/en/home/Products...
    Added: Thu May 02 2013, Hits: 4714, Reviews: 0  Write review   Cached
  • Theory, principles and background info Important Parameters of Quantitative PCR (qPCR) Analysis (Life Technologies)
    Very useful information about qPCR such as amplification efficiency, Setting the Baseline, Determination of Copy Number,Tm Calculations, PCR Optimization, etc.
    http://www.invitrogen.com/site/us/en/home/Referenc...
    Added: Thu May 02 2013, Hits: 3298, Reviews: 0  Write review   Cached
  • 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: 2091, Reviews: 0  Write review   Cached
  • Quantitative Reverse-Transcription PCR (q-RT-PCR) PDF file
    http://www.utexas.edu/research/sissonlab/protocols...
    Added: Sat Jan 31 2009, Hits: 17743, Reviews: 0  Write review  
  • 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: 1067, Reviews: 0  Write review   Cached
  • How to Reduce Primer Dimers in a LightCycler PCR PDF file (Roche Molecular Biochemicals)
    Offers optimization strategies to overcome primer dimer for real-time PCR.
    http://www.wzw.tum.de/gene-quantification/roche-pr...
    Added: Sun Sep 22 2002, Hits: 6782, Reviews: 0  Write review  
  • 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.wzw.tum.de/gene-quantification/adope-pd...
    Added: Sun Sep 22 2002, Hits: 7878, Reviews: 1 Read reviews  Write review  
  • Quantification using real-time PCR technology PDF file (Dr. Michael W. Pfaffl, Gene Quantification )
    The introduction of real-time PCR technology has significantly improved and simplified the quantification of nucleic acids, and this technology has become an invaluable tool for many scientists working in different disciplines. Especially in the field of molecular diagnostics, real-time PCR-based assays have gained favour in the recent past. However, the wide use of real-time PCR methods has also highlighted some of the critical points and limitations of these assays.
    http://www.wzw.tum.de/gene-quantification/klein-20...
    Added: Sun Sep 22 2002, Hits: 14472, Reviews: 0  Write review  
  • 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: 7272, Reviews: 0  Write review   Cached
  • Real-Time PCR Handbook (RRC Core Genomics Facility, University of Illinois at Chicago)
    Covers real-time PCR theories, methods, primer design, etc
    http://www.uic.edu/depts/rrc/cgf/realtime/start.ht...
    Added: Thu Feb 05 2009, Hits: 15507, Reviews: 0  Write review   Cached
  • 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.wzw.tum.de/gene-quantification/mackay-2...
    Added: Sun Sep 22 2002, Hits: 6202, Reviews: 0  Write review  
  • Real-Time PCR Quantification Using Cloned Standards and Multiple Housekeeping Genes
    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: Mon Feb 02 2009, Reviews: 0  Write review  
  • Sensitive mRNA Quantification - Gene Quantification Page (Institute of Physiology)
    Main focus of the GENE QUANTIFICATION web page is, to describe and summarize all technical aspects involved in quantitative gene expression analysis using real-time (kinetic) PCR & RT-PCR. e.g. detection chemistry, assay specificity, sensitivity, reproducibility, robustness, intra- & inter-assay variations, kinetic PCR efficiency calculation, quantification strategies, optimization strategies, data aquisition, data calculation, data normalisation, and statistics. To obtain high fidelity, accuracy and reliability in reverse transcription and subsequent kinetic PCR highly defined reliable quantification strategies, calculation procedures and statistical models are needed.
    http://www.wzw.tum.de/gene-quantification/index.sh...
    Added: Fri Jan 16 2004, Hits: 4919, Reviews: 0  Write review  
  • SYBR Green qPCR Protocol (Fero Lab, Fred Hutchinson Cancer Research Center)
    http://labs.fhcrc.org/fero/Protocols/Q-PCR.html
    Added: Sat Jan 31 2009, Hits: 13092, Reviews: 0  Write review   Cached