Using Real Time PCR for Cell Viability - Any tips? (Mar/23/2009 )
Because I don't want to waste reagents and materials, I would like to use TaqMan Real Time PCR to assess the cell viability on a 96-well plate. I figured that assessing for beta actin expression should probably be the most accurate. The cells tested will undergo knock-down and overexpression experiments, however I don't expect these to affect beta actin expression.
Does anybody have any experience and advice with this?
I agree mostly. However, some studies have pointed out the actin is not a good control and sometimes does change following certain treatment. Probably 18s or 28s rRNAs are better internal controls for rt-pcr.
If the control gene is not going to be affected by RNAi experiments, I think its expression can tell you the viability of the cells.
Thanks for your reply, I'll look into it. Perhaps GAPDH would be another option, also seeing we have the primer/probes for this gene. Alternatively, would amplifying a genomic region be another option, seeing that remains pretty constant in the cells?
Why don't you do a normal viability assay instead of wasting lots and lots of money on Taqman assays? You could do MTT, BCA, DNA binding etc.
I believe Baarsol is trying to save cost by obtaining cell viability data from expression analysis without performing additional assays such as MTT.
Amplifying genomic region may not tell you cell viability because dead cells may also contain the template.
Baars01 on Mar 24 2009, 09:37 PM said:
GAPDH is a good candidate as an internal control for qRT-PCR. Trust me, 18S and 28S RNA would save you lots of time. My personal opinion is that results of qRT-PCR was too indirect to demostrate cell viability change, especially when cell viability is a key issue in your research. If you simply want to use the result as an implication, it would be fine. Actually, MTT is very cheap and more convincible. May it help you and good luck.
Thanks for your responses,
I am familiar with the MTT assay. I should have mentioned that I intend to perform cell viability assays for cells that have undergone knock-down or upregulation of genes by transfection. The reason I would like to choose TaqMan as a cell viability assay is because I believe I would waste more money, time and effort in performing two transfections in parallel (one for qRT-PCR and the other for cell viability) for an MTT assay than using the same cDNA for two different qRT-PCR assays.
As two members here recommend the 18S and 28S RNA as good candidates I will probably go by those.