Measuring gene expression across cell lines
Posted 23 March 2010 - 06:28 AM
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.
Posted 23 March 2010 - 09:40 AM
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
Posted 23 March 2010 - 02:34 PM
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.