I extract RNA from about 30 000 cardiomyocytes with RNeasy Mini Kit(Qiagen), but I can not detect the concentration of RNA. However, GAPDH can be detected with RT-qPCR experiment. At the same time, I want to detect the relative mRNA expression level of a memberane receptor. I failed.
Any advice? Thanks.
Dear June,
If your GAPDH Ct value were within their best range.
Then, it quite possible that Primer (working stock) issue. Try freshly made primers.
Keep (positive) samples in which the membrane receptor expresses default.
I see here two potential problems:
1. memberane receptor is not expressed in these cells or the expression is very low.
2. the primers for the receptor are not working properly.
Dear Eugen@ Eugen Werwein and Saurabh@ Saurabh Mandal
Thanks for your reply.
Actually, the expression level of memberane receptor in cardiomyocytes is low(0.6%, relative expression to the reference gene GAPDH).
All the primers were freshly prepared and worked very well when I did RT-qPCR experiments with more than 200 000 cardiomyocytes.
With the same number of cardiomyocytes( 30 000 ), Ct value of GAPDH ranges during 19-23. So I think maybe the number of cells is too small and the RT-PCR kit is not good enough for such a small number of cells.
Dear June,
There is other way to think also you can check the isolated RNA conc., A280/260 values of your (200000) cardiomyocytes & cardiomyocytes (30000).
Also make sure, in cDNA synthesis process (something) should not wrong.
why I am saying, 30000 is quite enough to get RNA to do further experiments.
Definitely, RT-PCR kit efficiency should be kept under scanner.
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