qPCR for HKG and traditional PCR for GOI - (Jan/20/2006 )
I am screening the expression level of some novel genes (Gene of Interest GOI). For cost reason, it's better for me to use the traditional RT-PCR method for these genes but I have a good qPCR assay for housekeeping gene (HKG) and I was thinking doing a kind a mixed analysis:
Would it be valid to do
1) absolute quantification of the HKG
2) traditional quantitation of GOI (Et-Br gel, image analysis, densitometry)
3) computing the ratio density from GOI (2) by transcript number of HKG from (1)
Do you think that would be a valid approach ?
Thanks in advance,
based mostly on my instincts, I would say you are adding another complicated layer to your experimental design than is really necessary, and it would be very difficult to perform the appropriate controls to get your numbers nice and tight.
If you have a good 'traditional' RT-PCR method, I would think your best two options would be:
1. do only RT-PCR for your GOI and your HKG (I think I would do it this way)
2. perform an exhaustive set of parallel assays to make sure your mixed-approach is valid and will give consistent data...for example, do a bunch of samples both ways (the mixed assay with HKG and with GOI) and RT_PCR for your GOI and your HKG and see if you get the same results
for ease and sanity's sake, I would probably go with #1
the whole point of doing qPCR with your HKG and GOI in the same sample set is to validate your comparison of quantity, right? if you have to do densitometry for your HKG and qPCR for your HKG, what are you gaining by the mixed assay approach?