生物谷--分子生物学--技术

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导航：首页-->生物技术-->分子生物学技术指导（在线分子生物学技术方法）

RT-PCR
Competitive and Quantative RT-PCR
In Situ RT-PCR
RL-PCR
DNA Contamination
RT-PCR FAQ

RT-PCR
RT-PCR Protocol (UMBC)
First strand synthesis and subsequent PCR amplification. Based on LTI protocol
Reverse Transcriptase PCR (NWFSC)
Procedure for cDNA synthesis on dynabeads oligo(dT)₂₅
RT-PCR (Cause's Lab)
Detailed protocol for RT and PCR
RT-PCR (Lazo Lab)
First strand cDNA synthesis and PCR amplification
RT-PCR
Procedure either for RT-PCR and primer extension. For primer extension, just add hot dATP
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.
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.
Competitive and Quantative RT-PCR
Competitive RT-PCR (Dieter Kaufmann)
For quantifying mRNA, internal standard RNAs are added in a defined quantity to the RNA sample prior to the RT reaction. The resulting standard cDNA is coamplified with the same primers as the endogenous target sequence. Its PCR product is approximately 50 nucleotides smaller. This method allows measurement of small differences (as low as factor 2) in mRNA amount between RNA samples.
Competitive Quantitative RT-PCR (PDF) (Ambion)
Detailed protocol for competitive quantitative RT-PCR
Semi-Quantitative RT-PCR (Mike A. Dyer)
From RNA isolation, reverse transcription to PCR...
In Situ RT-PCR
RT In Situ PCR (Gerard J. Nuovo)
RT in situ PCR allows for the routine and rapid detection of low copy viral and human RNAs. Success with RT in situ PCR is best accomplished with formalin fixed, paraffin embedded material, which allows the study of archival material.
In Situ PCR On Plant Tissues (Bo Johansen)
Provides detailed protocol on tissue preparation, in situ PCR, detection and required reagents.
RL-PCR
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.
DNA Contamination
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.
DNAse I Protocol: The Use of DNAse I Prior to PCR (Invitrogen)
RT-PCR FAQ
How should I prime my cDNA? (Invitrogen)
How can I treat my RNA sample before RT-PCR to eliminate DNA contamination? (Invitrogen)
What is the highest temperature that reverse trascriptases can be used? (Invitrogen)
How can I tell if my RT-PCR product in RNA specific? (Invitrogen)
When is RNase H addition necessary? (Invitrogen)
How much of the first strand reaction should I add to the PCR? (Invitrogen)
RNase inhibitors and RNases (Invitrogen)
Troubleshooting Guide for RT PCR (Invitrogen)
General trobuleshooting for RT-PCR.