Hi there!
we are trying to measure the concentration of miRNA, after using miRNA extraction plasm/serum kit (Qiagen), by nano drop but the concentration is very low, so any one has an experience with that could tell us how we can measure the concentration? and what is the optimal miRNA concentration for expression study?
Hi Batool, this the best in my opinion you should avail for miRNA quantification;
https://www.thermofisher.com/order/catalog/product/Q32880#/Q32880
Because the miRNAs constitutes hardly 0.02% of all RNA species within a cell, and to measure it by nanodrop or any UV based protocol is not a proper method, as whatever readings you will get it is not purely from the miRNAs population and the actual miRNA content would be less from your observed readings, so ultimately the dye binding -based assays are quite specific for accurate quantification in this sense.
Regards.
hi Batool Ismail ,
miRNA are very less in the amount in total RNA plus they very small in size. So, better to analyze the level/expression of miRNA using qRT-PCR with miRNA primers.
After isolation, an important step is cDNA synthesis, the same Qiagen has miRNA specific cDNA synthesis kit which converts all miRNA present in RNA into cDNA. That cDNA can be analyzed using SYBr green dye.
Regards
Saurabh
For miRNA only, use any enrichment method to increase the final amount. Nano-drop is able to measure ng quantities but the peak will not be great. Better to confirm with RT-PCR
My suggestion is Qubit because it is more sensitive in comparing to nanodrop,
https://tools.thermofisher.com/content/sfs/manuals/Qubit_microRNA_Assay_UG.pdf
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