RT-PCR of different cell lines - (Nov/25/2004 )
This is the first time I am doing RT-PCR (reverse transcription pcr) and hence would really appreciate any sort of advice given.
I have 7 cell lines and i have extracted the RNA for all. I have obtained cDNA of these using Qiangen's omniscript RT kit and I would like to do a normal PCR to get an PCR product of around 240bp.
My question is: Do i need to optimise annealing temperatures for all the different cell lines? I am asking because I get different results for different clones of the same cell line!
Thanks in advance!
What do you mean by different results? Did you get different product size or different expression from those cells?
The annealing tempearture should be primer specific and has nothing to do with different cells. If the purpose of your RT-PCR is to determine the expression levels of your gene, once you have optimized the Ta for your primers you have to optimize the number of cycles so that the amplification will be in linear range. After you have cycled the PCR for that number of times, you may see band from some cells but not from other cells, which is possible because they may have different levels of gene expression.