For RT-Real time PCR, we have to take 1 sample produced without Reverse Transcription, as far as I know. I don't know too much because I am not yet finished, and I am going to validate the microarray result by qRT PCR.
For every gene(including referance gene) annealing tm should be same so you can do all genes rt expression at a same time in a single plate.
Hi Kumar, what do you mean with "no rt positive control"? Normally "no rt" is a negative control, and you will just need to run the same PCR you run on the rt on the no rt sample (with same dilution as your rt samples) and be sure you get a Ct at least +7 compared to your rt sample.
If you need a positive control, you can either use an rt which has already worked, or you can use a DNA fragment that includes your PCR target (could be an in vitro transcribed RNA, then you should retrotranscribe it like your sample, or just the DNA).
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