Hi Everyone:
I am studying a protein that is hypothesized to assist in transcription elongation. Its knockdown is expected to lead to decreased RNAPII processivity. I am trying to test this hypothesis by using qRT-PCR before moving to more laborious techniques, such as RNA-seq, GRO-Seq, etc. Basically, I am planning to use qRT-PCR to see if there is a decreased steady-state mRNA levels for promoter-distal regions in the knockdown cells, in comparison to wild type cells.
However, no previous studies have been done to this protein, so I don't know what reference gene I should use for normalization in qPCR. As this protein has been shown in other system to affect RNAPI transcription, I probably could not use Pol I-transcribed genes as well.
Any suggestions would be appreciated!
Hi Yang Zhang,
Why don't you identify the best reference genes for your specific interest? Try as much as candidate genes... Thus, you would help people who plant to study those genes. Moreover, you will have an extra publication.
If you can standardize or determine the amount of cells you analyse, you don't need an internal reference gene to normalize. If you can count but not adjust the amount of cells, you can use the determined cell number to normalize against.
You should make sure that the protocol is stable (quite constant [corrected] Ct values for replicated samples).
It doesn't matter if no one has done studies using this protein. You need to choose a steady-state "housekeeping" gene as your reference. Check what other folks have used in your same organism/cell line.
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