Sybr green RT-qPCR primer (View forum version)



rugs

Posted 14 June 2011 - 07:27 AM

hi,i am a newbie in the RT-qPCR. I will on study the gene expression of different interluekin genes by SYBRgreen RT-qPCR. I dont want to design the primers myself and i have found journals who have the primer sequence for those interleukin genes. However, these primer sequences are designed for taqman RT-qPCR analysis. Here, i would like to ask can i use those primer sequences (without taqman probe) and subjected the primers to sybr green RT-qPCR?

Besides that, one step RT-qPCR or two-step RT-qPCR is of better choice if i want to study lots of interlukine genes(around 15 genes) from a tissue sample?

Finally, can someone share with me on the flow of running the RT-qPCR? (A simple one will help me lots as i have read lots journal and i actually end up with confious)

pcrman

Posted 19 June 2011 - 08:51 PM

>>i have found journals who have the primer sequence for those interleukin genes. However, these primer sequences are designed for taqman RT-qPCR analysis. Here, i would like to ask can i use those primer sequences (without taqman probe) and subjected the primers to sybr green RT-qPCR?

Yes, you can. There are not many differences between these two types of PCR in terms of primer design.

>>Besides that, one step RT-qPCR or two-step RT-qPCR is of better choice if i want to study lots of interlukine genes(around 15 genes) from a tissue sample?

You should use two step RT-PCR especially if you have many genes to study.

>>Finally, can someone share with me on the flow of running the RT-qPCR? (A simple one will help me lots as i have read lots journal and i actually end up with confious)

Steps for qRT-PCR:

1. RNA isolation from tissues or cells
2. RNA quantification
3. RT reaction to convert RNA to cDNA, you will need: RNA, Oligo(dT) or random hexamer primers, reverse transcriptase and buffer, RNase inhibitor, dNTPs.
4. PCR reaction to amplify the resulted cDNA template, you will need: PCR primers, Sybr green mix, cDNA template. You will also need primers for a housekeeping gene such as GAPDH.