反向PCR

主要內容如下：

·         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 

·         cDNA Synthesis and Amplification of POLY(A) mRNA by RT-PCR (Immunology Resource)
CDNA synthesis from mRNA and subsequent PCR amplification 

·         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 situPCR is best accomplished with formalin fixed, paraffin embedded material, which allows the study of archival material.

·         In Situ RT-PCR (HYBAID)
Detailed Step-by-step protocol  

·         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 (LTI)

RT-PCR FAQ

• How should I prime my cDNA? (LTI)
   

• How can I treat my RNA sample before RT-PCR to eliminate DNA contamination? (LTI)
   

• What is the highest temperature that reverse trascriptases can be used? (LTI)
   

• How can I tell if my RT-PCR product in RNA specific? (LTI)
   

• When is RNase H addition necessary? (LTI)
   

• How much of the first strand reaction should I add to the PCR?(LTI)
   

• RNase inhibitors and RNases (LTI)