hi,

Isolation of RNA using TriZol is fine. But, if you are going with the Trizol method or even the miRNA isolation kit. You need to use miRNA specific cDNA kit for cDNA preparation. A regular cDNA kit doesn't do the job. I am considering that your miRNAs primers were synthesized well. My suggestion is to use ready-made primers from a standard company. Well, the ct value for the miRNA sample will be between that range only (ct 28-33). Even, based on the cDNA preparation kit, the qRT-PCR master mix varies. Many work with SYBr or some works with Taqman.

Regards

Saurabh

Are you concerned about the range of Ct values in your endogenous control? If your primers have efficiency that is 95-105%, then what you are seeing is simply variation in the amount of starting cDNA from each biological sample.

As Saurabh has said, you can't use a standard cDNA synthesis kit with random priming for qPCR of microRNAs. Think about the size of your miRNAs: they are only 21-25nt, so not much bigger than a primer, right? In order to do qPCR you need a template that is, at absolute minimum, large enough to place a forward and reverse primer on, so you need to use a reverse transcription strategy that makes miRNA-specific cDNA which is bigger than the miRNA to provide a binding site for a second primer.

How were the primer sequences you're using designed? If you got them from a paper, it's very likely that they are intended for miRNA cDNA made using stem-loop reverse transcription. The easiest way to understand this method is to google it and look at the diagrams that come up on Google Images. If this is the case, the paper probably also provides the sequences of the stem-loop primers that you will need to use for reverse transcription to capture the miRNAs you're interested in.

If you are using an elongation factor as a reference gene, you should not switch to an RNA extraction method that specifically isolates small RNA, because the mRNA for your elongation factor will not be present and your reference gene will not amplify. cT values between 26-30 are fine, and out to 35 might also be fine if you can identify a reason, for instance if you only input a small amount of RNA into the cDNA synthesis reaction.

I use the Mirvana Paris total RNA isolation kit following the miRNA enrichment protocol. We used several kits for isolation and had our genomics dept run each kit on the Bioanalyzer and this is best for miRNAs. For the RT-PCRs I use https://www.quantabio.com/products/microrna-profiling. Lots of helpful info on how to design your assay! In summary (you can read all the info) in cDNA generation, the kit elongates the miRNA with a poly A tail and the reverse primer is universal while the forward primer is basically the miRNA sequence. They have lots of links on QuantaBio including an excel of the forward primers and how to design them. They supply an internal control (SNORD44, similar to GAPDH in mRNA RT-PCRs) but I have better results with RNU6 as my internal control. Im studying sarcomas so my calibrator is U2OS. Im impressed with this method and after optimizing my protocol perfect melt curves and no primer dimers. Good luck!

Thanks a lot for sharing your experience and suggestions, Renee