How miRNA quality and quantity could be checked after total RNA extraction from serum either by kit method or trizol reagent?
Quantitative assessment of miR quality is usually done spectrophotometrically, and for qualitative assessment convert it into cDNA and go for an RT-qPCR assay with appropriate reference gene or spike in along with positive controls.
hi Javaid Wani
Generally, the amount of miRNA present is very very less in total RNA, possibly in serum or plasma, the miRNA content can be higher. But the amount of miRNA is not measurable by UV-absorption such as nanodrop. It is usually analyzed by qPCR.
Now, here the point is either you will use a miRNA isolation kit and convert those isolated miRNA into cDNA by taking desirable or preferable concentration OR you can isolate total RNA and using qiagen miscript RT II kit, you can convert all miRNA present in total RNA into miRNA-specific cDNA. That cDNA can be directly used for SYBr qRT-PCR. There you can use specific miRNA primers to analyze the miRNA level in that cDNA sample.
Regards
Saurabh
Hi, Javaid Wani and all others.
We always have a huge amount of options to do things. I did work with miRNAs in plasma from infected animals and humans and, in both cases, keeping the extraction from regularly the same amounts, presented very similar results by direct qRT-PCR.
If you need (or want to), instead of spectrophotometry, qubit fluorescent detection gives much better and reproductive results.
Any questions, feel free to contact me again.
Hops it helps.
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