thanks again for your answer.
If I’m able to get a signal from my miRNA in qPCR, is that not enough to confirm a successful isolation?
My plan is to isolate total RNA that contains miRNA with TRIzol. MiRNA analyses should be done with a commercial kit (first polyadenylation of miRNAs, cDNA synthesis and detection with SYBR green).
Even if a 1000bp sequence is isolated more efficient than a small one, the proportion should be the same in all analysed samples. Or am I wrong?
I just want to get the point why nobody is using one of the “other” genes for normalisation.
Some people are using TRIzol isolation in combination with columns. So these people only get small RNAs as the columns are made for this. In that case of course you can’t use a “normal” housekeeper and have to use a small RNA as control.
In my project I want to isolate total RNA including small RNAs from different tissue, so in theory I do have my “other” housekeeper for analyses. In contrast I also want to analyse miRNAs from plasma. There is no control and have to spike in something.
I personally don’t get the point why it is not popular to use a “normal” housekeeper if the analyse is made in tissue and total RNA is isolated.
I’m only searching for the right answer to understand that point. In my opinion there is in principle no difference. But may I’m totally wrong....
So thanks again for your help!