I did a qPCR of mammary tissue with two housekeeping genes (GAPDH and B-actin), but the Ct value is unstable among the control and the samples. Most of the Ct are around 21, but some of them are around 33. I repeated cDNA synthesis twice and I am pretty sure that I added the same amount of RNA in every samples. Also, I'm pretty sure that there is nothing wrong about pipetting and instruments.
Do you have any suggestions about internal control gene that I should use for the mammary tissue? Or what kind of control gene you ever used in rat mammary tissue?
There may be the presence of any inhibitors of amplification. I mean did you check the efficiency of your reaction? A wild guess though you may check eEF-2 or HPRT or RPS18. Nevertheless, the tool Genevestigator lets you know the best reference in your selected tissue based on many criteria like protein IHC or ICC or WB taking the data directly from protein atlas. Please try to follow MIQE guidelines if you really want meaningful data, otherwise, this whole experiment won't mean anything.
All the Best my friend from Iowa
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