I am using miRNeasy Serum/Plasma kit (Qiagen) to purify total RNA and I detected a low RNA concentration and low purity (260/280) using NanoVue. Bioanalyzer is the most recommended strategy to quantify circulating microRNAs, but it is not possible for me to use it.
After reading some articles about circulating miRNAs, I found no consensus about using a fixed RNA volume or a certain RNA quantity for reverse transcription.
Do you recommend using an input RNA volume or amount for RT?
Hi Sheyla
In my opinion you should follow a standard protocol for reverse transcription of miRNA pool....there are a lot of papers on this ...and if you are using a certain kit...follo the recommended protocol.....you may have a do a few trials initially....and once you know what is working in your hands go ahead and complete it...
The amount of RNA (or volume) you use for the RT-PCR reactions will vary according to the protocol recommendations, and other variables. However, in general, I recommend standardizing RT-PCR and PCR reactions with a known consistent amount of template, rather than standardizing by volume. If you just use the same volume of RNA template in each reaction, the actual amount, for example in ng, will vary from sample-to-sample. If you are doing anything quantitiative, this will result in unreliable and difficult-to-reproduce data. So, if possible, use the same actual amt of RNA in each reaction, assuming you have enough yield to assess yield and purity. Best of luck to you.
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