Hello all,
Can anyone please tell me how low can numbers go for mRNA expression of a gene when calculated relative to ActinB in mice? For example I have a gene with approx 0.08 relative expression and I am wondering if it has significantly high expression to investigate it further.
Thank you very much,
Diana
Hi Diana,
of course reproducibility has it's importance, but I assume your relative value is the result of an average of a significant biological sample number. If that is the case, I think I can see where you are going, if you push the system, probably qPCR could pick up also leakage expression or contamination or who knows what... Concerning this, I would suggest three considerations on which you can reflect a bit and then decide for yourself.
1) Actin mRNA is well known to be one of the most abundant mRNAs, if not THE most abundant, so any gene that is around 0.1 of actin mRNA (which is 1/10th) is essentially very highly expressed. You could easily accept up to 1/1000th and probably lower.
2) As a rule of thumb, an mRNA that has a Ct below 35, starting your RT-qPCR from 0.5 ug of cytoplasmic total RNA and a cDNA dilution of 1:10 should have a biological significance... assuming the reaction conditions are optimal, chemically clean RNA, no degradation, optimal primers (and probe if you use TaqMan or similar probes)
3) If your gene of interest is expressed at low levels, it is not wise to use actin mRNA to calibrate your reactions because the linearity of the PCR might not be possible for the two quantities, or at least you should calibrate the actin and gene of interest reactions encompassing the dilution you will use for the relative quantification and verify that for both you are in the linearity range (although you might have more than 10 cycles difference, for example...). In case you are not interested in finding specifically the relative amount of a set of genes to actin mRNA, use a less expressed gene for calibration and relative quantification between your samples, for example the TATA box binding protein.
Hope this helps finding your way. Good luck.
Hi Pietro,
Thank you so much for your reply it is indeed very helpful.
Kind regards,
Diana
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