I am currently using Exiqon's miRCURY RNA isolation kit for biofluids in order to isolate circulating mircoRNAs in rodent serum. I understand that small RNAs are but a small percentage of total RNA with microRNAs comprising an even smaller percentage of that which I have isolated. Normally when I run my isolated RNA from tissue on the Agilent Bioanalyzer I pretty reliably have RINs in the mid to high 8s, conversely running my isolated serum returns RINs in the realm of 2 to 3, and concentrations of around 70 to 100 pg. Should I be concerned that the small RNAs which I am isolating are degrading at a faster rate and therefore compromising my sample or is this a normal phenomena? Are RINs really all that reliable at that level? Also I'm currently using the RNA pico kit which may also have an effect.
I do not think RINs are relevant for quality analysis of serum micro RNA because the rRNAs are degraded. Is there a reference miRNA? Any thoughts out there?
As far as I know there are no equivalent reference miRNAs in rodent serum. When I run my array I will just normalize against the mean intensity of the total miRNA expression on the plate. I have had problems with amplification in a few individually assayed taqman PCRs so I'm wondering if it is just the quality of my isolated sample (degrades or just low yield) or just that my starting material is too low...I am inputting the maximum allowable volume per reaction of RNA in my RT and then preamping the sample afterwards.
Agree with previous posts. There is not much sense in doing RNA integrity analyses on serum/plasma (or other body fluids) derived RNA. However, we do assess the purity of the RNA by spiking in a synthetic miRNA. For normalization, we also use the (modified) global mean expression level, see my blog post on this subject.
http://www.biogazelle.com/how-find-stably-expressed-micrornas
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