We just started a new experiment regarding miR204 in my lab, & we want to see how much it is being expressed in our cell cultures. I tried extracting RNA using a PureLink kit from Thermo Fisher, measured it via Nanodrop, synthesized cDNA from it with a High-Sensitivity cDNA Synthesis Kit from Thermo Fisher, & ran qPCR on them with a program on BioRad CFX96.
However, there didn't seem to be any amplification for miR204, & somewhat-consistent but unexpectedly low amplification for 18S.
In your experience, what is the best cDNA kit for synthesizing cDNA from miRNA for use in qPCR? I want to make sure I'm not making technical errors in my extraction & analysis techniques.
I am a big fan of the advanced microRNA Assays from Applied Biosystems (Thermo Fisher). We use a mirvana RNA/microRNA kit, then amplify using the ADVANCED microRNA cDNA kit. The kit works with a poly a tail, so that depending on the Assay being used (mir 204 in your case), you can measure ANY microRNA present. The Kit amplifies everything that is there, so that also in teh future, other mirnas can be measured from the same cDNA. Just my experience.
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