2 replies to this topic

[super_novice]

Hello

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?  

Please help!!!!

Super_novice

[AlessandraCastaldi]

Hi,

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.

Good luck!
alessandra

[Neanderthal]

Please see the link below:

http://www3.appliedb.../cms_044972.pdf

A list of controls are mentioned in this PDF. Hope it helps.

Thanks,

K.