Overview: 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.
Subcategories
Related Categories:
Protocols
- Gene-specific RT-PCR
(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: 14985, Reviews: 0 Write review -
Complete RT-PCR Protocol
(Gumbleton Lab, Cardiff University)
From RNA isolation, quantification to RT reaction, PCR and gel visualization.
http://www.cf.ac.uk/phrmy/PCB/LabProtocolsPDFs/rtP...
Added: Fri Jul 20 2007, Hits: 5557, Reviews: 0 Write review -
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: 3215, 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: 4084, Reviews: 0 Write review Cached -
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: 6254, 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: 1291, 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: 3646, 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: 1095, Reviews: 0 Write review Cached -
Reverse Transcriptase PCR
(NWFSC)
Procedure for cDNA synthesis on dynabeads oligo(dT)25
http://micro.nwfsc.noaa.gov/protocols/revtrpcr.htm...
Added: Tue May 14 2002, Hits: 5198, Reviews: 0 Write review Cached -
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: 11301, Reviews: 0 Write review Cached
Trouble Shootings and FAQs
- PCR contamination in negative control
- Which gene is the best internal control for RT-PCR?
- How to know the primers span an intron within amplified region?
- Tips on first strand cDNA sythesis with high GC contents
- RT-PCR negative control shows the correct band
- Tips on primer design for RT-PCR
- Troubleshooting RT-PCR
- RT-PCR, why two PCR steps?- two PCRs after reverse transcription?
- Should I dilute RT reaction prior to PCR?
- Why is DTT used in reverse transcription (RT) reaction?
- RT-PCR basics
- How to produce a high-quality cDNA template for RT-PCR?
- Tips on RT-PCR for intronless genes
- RT-PCR primer design guide- How to check gene structure and design the primer?
- How to synthesize your own oligo-dT primer-