Hello, I have a research thesis in the field of measuring the expression of several genes in PBMC cells in patients with breast cancer, I extract RNA by using the phenol/chloroform protocol, and take 2 ug of RNA after the DNase treatment for cDNA synthesis (the maximum amount stated in the protocol (2ug)) after synthesis of cDNA, add 5 uL of injection water to the final volume of cDNA (20 uL) and use 1 - 3 uL for q-PCR, but Ct for 18s gene (internal-control) is about 25, which is a high number, (while my friends using the same kit of the same company and the same cell source get a lower Ct around 14 to 9) Is there a way to reduce the Ct and increase the quality of cDNA? Should I use less RNA for cDNA synthesis?
Can you show us your Ct curve(s), I do think that you should be getting 18S around 15-20 Ct. Seems like your input is pretty high for cDNA synthesis to make that happen. Ask your friend to analyse your sample with his PCR consumables as well, if he gets late Ct then we know something is different with your sample.
In my opinion 1-2µg of total RNA input for cDNA synthesis is ok, but the input for qPCR is very high. Usually we dilute the 20µl cDNA 1:5, and of these 100µl, take 2µl as input for qPCR. Adding more template does not necessarily result in lower CT values.
And make sure that during phenol/chloroform RNA extraction you do not get any traces of phenol into your RNA, this will inhibit any downstream enzymatic reaction.
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