I am aware that 25-50 ng/reaction of total RNA are sufficient to run a qPCR and that mRNA only makes up a small percentage of total RNA. I have extracted and purified mRNA for experiments and was wondering how much mRNA (ng/rection) would be required for successful qPCR analysis.
Dear Dr. Walsh, I always use approx. 200 ng/reaction total RNA for qPCR. I think it is suitable for you reaction. But it depends on your protocol. Some protocols need 200ng total RNA per reaction but the others need less.
It's depending on your reference and target genes. You should optimize the concentration accordingly. It is also depending on the final concentration which can be affected by the concentration changes resulted from the cDNA synthesis. A few ng per microliter is adequate for the final concentration
Yo dieter, my routine qPCR involves 4ug total RNA in which after reverse transcription (made into cDNA), I further dilute them in 125x / 25x. Also, it depends on the qPCR machine and reagent mix you use. In my case, I use KAPA SYBR Green kit and with Applied Biosystem Vii7A RealTime machine. Therefore, the smallest concentration of cDNA I have used is ~140ng in 4.4ul in which the CT value for housekeeping genes would be around 22 (in mammalian cell lines).
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