miRNA qPCR normalization/control gene sequence!!!! - (Oct/17/2012 )
I am absolutely new to the area of miRNA expression quantification.
I quantified miR-122 and miR-370 with U6 snRNA as the normalization control using qPCR (Roche SYBR Green and Lightcycler 480).
The miRNA cDNA synthesis kit was from SABiosciences.
The primer sequences that I used for all three were from peer reviewed publications.
However, the U6 control had a huge variability in my biological replicates whereas the actual test genes were not bad at all.
Also, the U6 Ct values were higher than my test genes indicating a lower abundance in my samples.
My questions are:
1. Can someone suggest me a few control miRNA genes that I can use for these quantifications? I am using liver tissue.
2. If possible I would like the sequences of these control gene primers too
3. Any suggestions about the techniques I am using?
4. Should I be using cDNA and the qPCR kits from the same company?
I use to quantify microRNAs by EXIQON kit (mircuryLNA), that, as far as I see, is one of the best. You can use other endogenous controls such as 5S, RNU5G, RNU1A1, that are small RNA. I'm sorry but I don't have sequences since I use to buy them directly from EXIQON, but I'm sure you can find them somewhere. However, I would sugest to give a look on EXIQON website, even if you are not using their kit, there are good sugestion about miRNA quantification.
Please see the link below:
A list of controls are mentioned in this PDF. Hope it helps.