General idea about RT-PCR - RT-PCR (Apr/04/2006 )
I have no idea about RT-PCR, would anyone please inform me about this experiment?
I would like to know about the procedure and use of RT-PCR, and about the available kits in the market.
If you have some cool web links you can aso add that to your email.
Thanks in advance
RT-PCR stands for Reverse Transcriptase Polymerase Chain Reaction.
simply, it is used to amplify complementary DNA strands from RNA precursors. A simple protocol involves isolating RNA fractions (normally mRNA) then denaturing before adding the enzyme and dNTPs and a 3' primer - either oligo(dT) or random primers. The reverse transcriptase is a viral enzyme which uses the RNA molecule as a template for the synthesis of a complementary DNA strand. This process is called First Strand Synthesis. You are left with single-stranded DNA.
This ssDNA can, in fact, then be used directly in PCR to amplify the cDNA. It is also possible to synthesise a second strand of cDNA to give you dsDNA which can then be used to create cRNA (via an RNA polymerase) which can be used in microarray studies.
Firt things first ... Are you asking about RT-PCR or RealTime PCR (other abbreviations qPCR, qRT-PCR). It's a common problem. RT-PCR is a Reverse Transcriptase Polymerase Chain Reaction and Doc Martin described this method. Lot of people by mistake use the same name for RealTime PCR.
In a shortcut - RealTime PCR is a hybrid technology which connects classic PCR method with spectrofluorimetry. Thanks to addition of dsDNA binding fluorophore (SYBR Green I for example) or fluorogenic hibridization probes to PCR mix you can observe accumulation of your PCR product on computer screen (plot of fluorescence change during cycling).
RealTime PCR is a powerful method of DNA/RNA quantitation, genotyping, SNP analysis and gene expression research