PCR or RT-PCR? - (Sep/22/2004 )
I'd really appreciate if someone would clarify this for me. I use RT-PCR for quantitfying the levels of gene expression in my samples but if i just want to know whether the gene is there or not can I use PCR?
Does PCR pick up the gene regardless of whether it is capable of being transcribed to RNA, i.e. a working gene.
Just wondering have I been wasting my time with the RT steps.
please correct me if I misunderstand your question.
RT-PCR is used for measuring message,you are quite right. The gene shouls almost always be there, unless you are talking special cases, like the Y chromosome, or something, so normal PCR would come up. So go with RT-PCR unless you are working with special cases, or you ar eworking with transfected plasmid DNA- then normal PCR would be ok, as long as you work wuth plamid-specifi, rather than gene specific primers (that could pick up endogenous gene).
Hope that answers your question
I Don't quite understand what you really want to know. You want to check if your gene locus is deleted (such as LOH) or if your gene is expressed, which one? If the answer is the formmer, you should use genomic PCR or SSCP; if the latter, use RT-PCR.
PCR should be alright if you are not interested in ascertaining whether there is a change in expression.