Measuring gene expression across cell lines - (Mar/23/2010 )
I am trying to measure the expression level of a particular gene across 6 different mouse cell lines. The relative levels of expression are consistent with what is expected however, the absolute values are coming up as less than 1 copy (up to 100-fold less) per cell, and this is not consistent with expected values.
One thing I did that showed an increase in detection of the target gene was to use a gene specific primer, as opposed to random hexamers, for synthesis of cDNA. Despite about a 10-fold increase in detection by using this modification, the absolute values are still low.
Are there any suggestions as to how else I might try to optimize this assay? I am using Qiagen's RNeasy Plus Mini isolation kit. RNA quality is fine with 260/280 averaging at 2.06. I am using ABI's reverse transcription kit and Universal PCR master mix, with custom designed Taqman probe/primers.
Also, what is the industry standard, if there is any, for housekeeping genes in mouse cell lines? I am using GAPDH because this was a readily available endogenous inventoried primer/probe set for mouse supplied by ABI.
Measuring gene expression by qRT-PCR is new to me. I've done plenty of viral load work with qRT-PCR but this is very straightforward and relatively problem free.
Thank you for any suggestions you may have to offer.
have you checked the location of the primers? maybe they are detecting just a low abundant splice variant? why do you expect this gene to be higher expressed?
how does the GAPDH expression between your cell lines look like? Usually, the "classical" reference genes can give quite consistent results between different cell lines with exceptions being the rule
I think tea-test raised a good point regarding why you got very low Ct value for your gene. Check the primer location to see which region it is targeting.
Regarding housekeeping genes, gapdh is not a good control becuase it's expression is so aboundant and it has many pesudogenes in the genome. Even validated primers from ABI may not necessarily be good controls. If you use another housekeeping gene, most likely you will get different results.
Other than that, I think you are doing fine.