Hello,
I'm studying for a expression of microRNA in MOUSE granulosa cell.
I have used miRCURY LNA miRNA PCR Starter kit for cDNA synthesis and real-time PCR
While i was searching for the process, I would like to know which gene should i use for normalization?
Because miRNAs are so short that they may have very different behavior during extraction and reverse transcription than longer transcripts. Depending on the sample’s origin, i have to use different gene for normalization.
I have found some of gens such as snoRNAs and snRNAs including U6, but i'm so confused which one is match for my sample.
Could you please let me know the gene is good for normalization and why?
I know U6 is traditional, but I would honestly suggest using another miR (or ideally two or three). Apply the same rationale to miR work as you would to mRNA work, and try to identify stable reference miRs. Consult the literature for possible stable candidates in your biological system. I use miR23a and miR223 for serum miR work, for example.
One cannot understate the importance of validating reference genes as it is often an overlooked practice. You will need to determine this by conducting a reference gene stability test by picking several genes and use a software (i.e. genorm) to determine its stability. Stephen Bustin has good articles and seminars about this and i refer you to one of the most important articles in this field: Article The MIQE Guidelines: minimum Information for Publication of ...
I used u6 RNA to normalise.I have came across publication where they used specific mirna itself as normalised provided that it does not get affected by ur treatment
Hi I used u6 as normaliser.but u can use any other also provided that will not be impacted by the treatments ur using
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