Hey Parwez,

What do you mean by 'significant' Ct values?

Have you tested the primers on any standards such as cell lines? It could be that these miRNAs are not (or low) expressed in your samples.

Regards,

Siva

Hi Sivanesan,

Thank you for your answer. Significant Ct values just mean not undetermined. Actually, after miRNA genechip analysis data, we got miR-6089 as our endogenous control for our validation phase which is not found in normal TaqMan assay, so we ordered Advanced. But, its not working in my case in FFPE sample even with quite good concentration of RNA.

Regards,

Parwez

What method are you using to extract your microRNA? miRVana, Qiagen kit for miRNA or TRIzol?

Hi Pedro,

I used miRVana.

Well, in this case, to obtain a sample suitable to do qPCR, you don't need to do the protocol of extraction until the phase of enrichment. I made this mistake in my first experiments also. And I was having the same problem, but with a different kind of sample (kidney). I asked help to ambion scientific advisory, that told me to do the extraction only until the total RNA phase of the protocol of miRvana kit.

To be more precise, in the figure 1 at the page 2 of the manual of the kit (https://tools.thermofisher.com/content/sfs/manuals/fm_1560.pdf) , instead of continue the extraction making the steps described in the bellow (small RNAs arrow), simply do the steps described in the upper part of the figure (total RNA arrow).

I hope that It helps you.

Pedro

Thank your very much for kind suggestion.

I used the miRNeasy serum / plasma kit for circulating miRNAs