RT-PCR Links & Info: Get the Facts

[Goodie]

links and info of RT- PCR?

appreciate for any inputs.

 

[Monique]

First link that comes up in a google search, gives a good explanation http://www.ambion.com/techlib/basics/rtpcr/

 

[Goodie]

As i know RT-PCR means Real Time PCR and Reverse Transcription PCR. 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?

 

[Monique]

Both: it's real time RT PCR

Reverse transcription means that you make cDNA out of an RNA template. For quantitative analysis you need to use real time PCR.

So you do reverse transcription PCR, and you watch with every PCR cycle what your cDNA quantity is (real time). At the same time you do reverse transcription on a sample of RNA that you know the concentration of. You can relate the two samples and find out the concentration of the unknown.

 

[iansmith]

As i know RT-PCR means Real Time PCR and Reverse Transcription PCR. 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?

RT-PCR mean Reverse transcritpion PCR. This is a convention and anybody telling that it mean Real Time PCR or both is confused about the terms. For real time PCR, the word quantitative is added to it. So you can have quantitative PCR using cDNA library or just DNA. You can also do one step quantitative RT-PCR.

A one step RT-PCR is qualitative. It will allow to determine if the transcript is present or absent. However you will not be able to determine if the level is different if it present in both samples.

For Real-time, you require some specific equiment with specific reagent. If you lack the equiment, then there is an alternative called semi-quantative PCR. the idea is to run a normal PCR either using a dertime amount of cDNA or DNA but stoping the reaction every five cycles.

For example, you run your RT-PCR and the you start you PCR step. After 10 cycle you remove you sample, resume the program and remove a sample every 5 cycle until you reach 30 cycles. You then analyse each samples on a gel and see when your band occurs. In one sample it might take 25 cycle for the band to occus whereas the other sample appears after 15 cycle. You can also measure the intensity of band from the picture. However, personnaly I find that the intensity is arbitrary and you need a good difference between bands to make a solid statement.

the difference between quantitative and semi-quantive is that quantitative will tell you the exact cycle (ex: cycle 13) when your sample passed the threshold limits whereas the semi-quantitative will give an estimate of the time the sample pass the threshold limit (ex: between cycle 10 and 15).

For your experiment, you first need to do an RT-PCR followed by PCR so can determine if the transcript if present or absent in your desired conditions. Semi-quantitative and quantitative might then be required to determine if there any significant difference between level of transcript.