I was struggling with the standard curves for my RT-qPCR, and my PI suggested trying one-step instead of 2-step. After diluting one of my RNA samples to generate a standard curve (started with ~200ng/ul) 6 log dilution series, I tested my primers, and it worked wonderfully. (SYBR green one-step supermix)
So now I have partitioned enough RNA from all my samples into one master RNA mix (~400ng/ul) and created one large dilution series to use for my standard curves on all my plates.
My question is: Do I have to dilute all my RNA samples to the same concentration? Or can I just dilute them all 1:100 to get them into the middle of the standard curve range? The concentration of the RNA samples ranges from 50ug/ul to 1000ug/ul.
I have 2 genes of interest that I am going to run, and 2 housekeeping genes for normalization. I have 54 samples, and will need to run 3 plates per gene to get all the samples through. (6 wells of standard curve*3, one NTC*3, and the remainder of the wells for samples.)
Is there anything I am missing here? Thanks for looking!
Austin
Hi
There is no need that all samples are of same concentration, but for optimal quantification they should have Cq values between 20-30. Different dilution would also complicate further calculations. Good luck!
Spela
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