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internal control in RT-PCR - inquiry (Jul/08/2011 )

Hello All!
I hope to ask about using internal control in the RT-PCR technique (reverse- transcriptase PCR)

what is more suitable?
using the internal control primers in the same tube with the tested gene primers as (Multiplex PCR reaction)
using internal control primers in a separate tube and then how could I normalize it with my tested gene???



It is better to run the internal control or housekeeping gene PCR separately. If you use the same amount of RNA in RT reaction for all samples and then equal amount of cDNA as PCR template, you can then use what you get from the control gene to normalize your target gene.


as pcrman kindly said : separately ...
the way we do it :-
1) RNA extraction.
2) measuring conc. in order to start with same conc.s of samples in the run.
3) RT.
4) housekeeping gene detection. ( internal control )
5) along with detecting our gene of interest.