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Real Time PCR or Reverse Transcription PCR?

Discussion of all aspects of biological molecules, biochemical processes and laboratory procedures in the field.

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Real Time PCR or Reverse Transcription PCR?

Postby chancer » Sun Mar 20, 2005 1:09 am

When i want to examine up or down regulation of my gene expression which one should i use; Real Time PCR or Reverse Transcription PCR?

Thanks.
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Postby zerotimbo2001 » Wed Mar 23, 2005 4:18 pm

Hi

Actually real time PCR commonly written as RT PCR is in fact QPCR because you are quantifiying your replicons in real time. this is just a techinque

RT PCR or reverse transcription PCR is what you want. Firsty extract your RNA, use TRIZOL it gives better recovery than nucloespin columns, secondly you really should have at 1.9-2.0 quality or your samples will be degarded somewhat. Make sure you have the correct primers. When findng wether up/down regulation RNA expression is what you want remember RNA equals Protein as the end product (very loose statement but you get the drift)

Once acheived run your sample down a gel and see wether you have RNA, when you do start the reaction I use the BEAD set up, i.e little eppendorfs with alll your reagents in it TAQ etc.... (amersham do them) then wip it into a thermo cycler PCR it and you probably know what to do after that...

Would also be nice to do some 2D proteomics on your protein sample as well cos you get to kill two birds with one stone, you can look for up/down regulation and also elucidate novel protien production.

Have fun
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Postby DevGrp » Thu Mar 24, 2005 4:44 pm

It depends on what you want to find out.
Standard RT-PCR (reverse transcriptase -PCR followed by an agarose gel of your products) will tell you if your gene is expressed / not expressed under your experimental conditions. It's also ok at telling you if the expression goes from lots to very little or the reverse. It is really a qualitative technique "is my gene expressed yes/no?"

For any changes more subtle than this you probably need qRT-PCR "quantitative reverse transcriptase-PCR" aka realtime RT-PCR. There are various ways of doing this. The original uses Taqman probes and is sold by Applied Biosystems. There are now various other technologies. The problem with all qPCR is that the cost soon adds up, although if you don't get your reagents from applied biosystems it's getting cheaper!
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