I am working on U87 & U251 cell lines. After treatment in triplicates isolated total RNA to cDNA. While doing real-time PCR can I use the pooled cDNA from triplicates as reference sample for each experiment or/and can I use the same for all other real-time PCR experiments. Can you please suggest any other ways?
yes you can. We always isolate cDNA from rat tissue that is known to express the RNA that we are looking to amplify. For instance, when we are looking at AT2 receptor RNA levels in rat adrenal glands, we will take kidneys from those rats and isolate a ton of rat kidney cDNA. We use this pooled cDNA to run our PCR curve so that we do not have to waste cDNA from the tissue that we are specifically looking at. This way you will have plenty of cDNA to perform multiple standard curves to always determine the efficiency of the PCR reaction.
Yes I concur. Infact, because of natural variation between technical cDNA replicates reference literature will actually tell you to set uo between 3 and 5 replicates (reactions) pool and then aliquot for styorage purposes
I am working on clinical samples of control pateint and want to study copy number variation by using Real Time PCR. I cant understand what to choose as reference sample.Please help me.
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