Top : Molecular Biology : PCR : RT-PCR

Summary: RT-PCR (reverse transcription-polymerase chain reaction) is a sensitive method for the detection of mRNA expression levels. Traditionally RT-PCR involves two steps: the RT reaction and PCR amplification. RNA is first reverse transcribed into cDNA using a reverse transcriptase as described here, the resulting cDNA is used as templates for subsequent PCR amplification using primers specific for one or more genes. RT-PCR can also be carried out as one-step RT-PCR in which all reaction components are mixed in one tube prior to starting the reactions. Although one-step RT-PCR offers simplicity and convenience and minimizes the possibility for contamination, the resulting cDNA cannot be repeated used as in two step RT-PCR.
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Protocols
  • First strand cDNA synthesis PDF file (Practical Approach Online, Oxford University Press)
    Detailed protocol of synthesizing first strand cDNA for RT-PCR or other applications
    http://www.oup.co.uk/pdf/pas/5-2-2.pdf
    Added: Sat Aug 17 2002, Hits: 17919, Reviews: 0  Write review  
  • Gene-specific RT-PCR PDF file (Molecular Profiling Initiative, NCI)
    This protocol is used for microdissected RNA, but may apply to other situations. It introduce three three priming methods for generating first strand cDNA from mRNA: 1) Oligo (dT)-based priming is preferred when the RNA is of high quality, since first strand polymerization should be continuous from the 3' end of the transcript to the desired sequence regardless of the length of the 3' UTR. 2)Random hexamer priming is recommended for fragmented mRNA (lengths <500 bases)where there may be discontinuity between the desired sequence and the poly A tail. 3)A primer that is specific to the gene of interest is best when amplifying low abundance transcripts.
    http://cgap-mf.nih.gov/Protocols/PDFFormat/GeneSpe...
    Added: Sat Aug 24 2002, Hits: 13088, Reviews: 0  Write review  
  • Reverse transcriptase (RT)-PCR PDF file (Langdale Lab, Department of Plant Sciences, University of Oxford)
    Protocol describes DNase treatment, the RT reaction and PCR.
    http://dps.plants.ox.ac.uk/langdalelab/protocols/P...
    Added: Wed Feb 11 2009, Hits: 23238, Reviews: 0  Write review  
  • Standard RT-PCR (Pamela Stanley Lab Wiki, Yale University)
    RT-PCR or reverse transcription PCR refers to PCR that uses product of an RT reaction as template. In effect, the PCR amplifies cDNA fragments. In one-tube RT-PCR, RNA and PCR primers are added to a reaction mix that is thermocycled for RT first followed by for PCR. One-tube RT-PCR reaction mixes are supplied by many manufacturers. Drawbacks to their use include lack of flexibility. Otherwise, RT-PCR is a two step process.
    http://stanxterm.aecom.yu.edu/wiki/index.php?page=...
    Added: Fri Mar 20 2009, Hits: 18378, Reviews: 0  Write review   Cached
  • Detecting DNA Contamination in RT-PCR (Qiagen)
    To avoid Detecting DNA contamination in RT-PCR, use controls, design approporiate primers.
    http://www1.qiagen.com/resources/info/detecting_dn...
    Added: Sun Jul 21 2002, Hits: 3826, Reviews: 0  Write review   Cached
  • DNA Contamination in RT-PCR (Ambion)
    Present data showing levels of DNA contamination in RNA generated by different procedures and suggest several precautionary measures which can be implemented to reduce the impact of this persistent problem.
    http://www.ambion.com/techlib/tb/tb_176.html
    Added: Tue May 14 2002, Hits: 3023, Reviews: 0  Write review   Cached
  • First Strand cDNA Synthesis PDF file (Practical Approach Online, Oxford University Press)
    http://www.oup.co.uk/pdf/pas/5-5-2.pdf
    Added: Sat Aug 17 2002, Hits: 4790, Reviews: 0  Write review  
  • Gene-specific RT-PCR (Cancer Gene Anatomy Project, NCI)
    Protocol may be used for amplifying individual transcripts from RNA recovered from microdissected cell populations. The procedure for reverse transcription is as regular RT, but for PCR, radioactive dCTP is used and PCR products are separated by running polyacrylamide gel.
    http://cgap-mf.nih.gov/Protocols/DNARNAProteomicAn...
    Added: Tue May 14 2002, Hits: 666, Reviews: 0  Write review   Cached
  • Nested RT-PCR From Paraffin Section (Hans Popper)
    This protocol is for the non-isotopic detection of hepatitis C RNA and albumin mRNA (as an internal control) from 4 micron sections of formalin-fixed, paraffin-embedded liver biopsies by RT-PCR. The procedure for RNA extraction from formalin fixed paraffin embedded section and PCR amplification is described.
    http://zapruder.pds.med.umich.edu/users/Frank/HepC...
    Added: Tue May 14 2002, Hits: 299, Reviews: 0  Write review   Cached
  • Preparation of Template RNA for RT-PCR (Roche Diagnostics Corporation)
    Guidelines for preparing high quality RNA template for RT-PCR.
    http://www.roche-applied-science.com/pcr/applicati...
    Added: Sat Sep 21 2002, Hits: 779, Reviews: 0  Write review   Cached
  • Reverse Ligation Mediated RT-PCR (Antisense Research Group)
    RL-PCR can be broken down into a number of simple steps. Synthesis by in vitro transcription and purification of an RNA linker species. Extraction of total RNA from cells into which oligodeoxynucleotide has been delivered by, for example, streptolysin O permeabilization. Ligation of the RNA linker to all available 5' monophosphates in the purified total cellular RNA sample. Reverse Transcription of the RNA using a gene - specific primer. Amplification of specific fragments by PCR using linker - specific and gene - specific primers. Sub- amplification of the first PCR product using linker specific and nested, labelled, gene specific primers.
    http://www.liv.ac.uk/~giles/methods/rlpcr.htm
    Added: Tue May 14 2002, Hits: 1752, Reviews: 0  Write review  
  • RT-PCR (Lazo Lab)
    First strand cDNA synthesis and PCR amplification
    http://wheat.pw.usda.gov/~lazo/methods/lazo/revtrp...
    Added: Tue May 14 2002, Hits: 3896, Reviews: 0  Write review   Cached
  • RT-PCR procedures (Roche Diagnostics Corporation)
    RT-PCR can be performed as either a two-step (one tube or two tube) or an one-step (coupled RT-PCR) procedure. This protocol introduces both methods and their advantages.
    http://www.roche-applied-science.com/pcr/applicati...
    Added: Sat Sep 21 2002, Hits: 1445, Reviews: 0  Write review   Cached
  • RT-PCR Protocol (Julie B. Wolf, University of Maryland, Baltimore County)
    First strand synthesis and subsequent PCR amplification. Based on LTI protocol
    http://www.research.umbc.edu/~jwolf/m12.htm
    Added: Tue May 14 2002, Hits: 8225, Reviews: 0  Write review   Cached