Dear Parul , using of single reference gene it is not a good idea. Please try with at least 2, better 3 in one experiment. before it the best way is to determine the most stable genes in your experimental conditions.

Dear Parul,

Regarding normalisation of miRNAs data using U6 there is a lot of publication against the use of U6 as a normalizer whatever the material you use. I invite you to read miRQC paper from Mestdagh (2014) you should find a lot of good advices in your miRNA research on plasma. Have you already check for stably expressed miRNA into your experimental plan? Some publications proposed hsa-miR-16 or hsa-miR-23 as stable miRs. You may not use spike-in if you use some to check both RNA purification (cel-miR39) or RT efficiency with Qiagen protocols....

Are you using miRs panels? what kind of product (Taqman, miRCURY or miScript?).

Looking forward to receiving your return.

best.

Arnaud

@ Arnaud Beurdeley

Thank you for your suggestions. I am using miScript for cDNA and RT reactions. I am actually working on bovine plasma. So do you think in this case I should use hsa-miR-23?? Although I have used bta-miR-23 which is stably expressed in my experimental plan.

@ Andrei S. Babenka

Thanks for your reply. I will surely look to include more than two reference miRNA in my experimental plan.