QPCR versus regular PCR for Chip - (Nov/01/2007 )
My project is to examine whether a transcription factor binds with the promoter region of a target gene. So, it's basically a "yes" or "no" question. While I read the papers, I saw a lot of people are using regular PCR (agarose gel and EB detection) for Chip detection . But the other lab in our deparment is using QPCR for all of their Chip experiments and it looks like that everyone in this forum is also using QPCR.
So, I am wondering whether I can use regular PCR instead of QPCR for my purpose . Regular PCR are more convenient and cheaper than real time PCR in our lab, because our lab don't have QPCR machine and have to pay a lot money to let a genomic facility to run the QPCR for us.
Semi-quantitative PCR can be used for ChIP.
The only drawback is that it is not quantitative and
if you do not get an "on/off" answer it is more confusing than QPCR.
The impt. parameters for semiquantitative are as follows:
#1 Make sure your primers amplify in linear range. Do not Saturate pull down products!
#2 ALWAYS USE NEGATIVE CONTROLS.
#3 ALWAYS USE POSITIVE CONTROLS.
#4 Did I mention positive and negative controls?
When doing semiquantitative the results are usually clear when your bands
are compared directly (on the same gel) to positive and negative controls.
for ChIP I do both quantitative and "normal" PCR. Normal PCR is to check whether I have signal or not and whether my inputs are in equal amount. If I am comparing the amounts of DNA (i.e. in a time course experiment), then I do real-time PCR as well. There I can normalize my samples to the inputs and express amount of samples as fold-change. In normal PCR, it is also possible that I have a lot DNA as starting material and the signal reaches a plateau (then it wouldn`t be possible to compare my samples). There I use real-time PCR as well.