Hi! The best way is to test several miRNA and check the most stable ones by Normfinder or Genorm software; by the way, in my plasma samples mir-103a-3p and 93a-5p, were the best choices. Important, i'm not working with cancer samples, depending on the pathology microRNA can have different expression levels and stability!Another method which is very useful with plasma and serum where a proper housekeeping is hard to find, is the global mean if you have several samples (not only 2-3). This means that you can run your Realtime without the housekeeping and normalize the ct value of each sample on the global mean of your experiment for that specific microRNA.
Hope it helps
Claudia
You can use miR-99a-5p and miR-139-5p as control in serum due to their consistency across all sample sets. As a reference for this information;
http://www.biomedcentral.com/1472-6890/14/27
I usually use spike-in controls.
I add two spike.ins to the simple prior to miRNA extraction (cel-34 and cel-238) and a spike-in control to the RNA simple prior to the reverse transcription reaction (cel-54).
When adding the spike-in controls to the simple before extraction, be sure that you add them after the addition of the lysis buffer or the phenol (dependidng of the extraction method) to avoid degradation of the spike-in control sequences.
Regards
Thank you all, I will use spike-in. But wouldnt there be a difference between a protein bound native miRNA and unbound spike-in miRNAs in practice? After all protein bound miRNA should last longer and harder to isolate. btw, I plan not to use any reverse transcription, instead using isolate from serum directly.
Lidia,
What kit did you use for the miRNA extraction?
Do you add the spike-in twice to your sample (before the lysis and before the RT)?
What sample do you use to normalize you reaction?
Hi Mariana,
I use the miRNeasy mini Kit (Qiagen) for cells and miRCury Extraction Kit (Exicon) for biobluids.
For cells, I use RNU6, RNU1A and RNU43 as references
For biofluids,
I add two spike-in controls (cel-miR34 and cel-miR238) after lysis buffer and 1 spike-in control before RT (cel-miR54)
I use more tan one reference spike-in in each PCR and select the best for normalization.
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