4 replies to this topic

[mcb]

Has anyone experience with the miScript (Qiagen) to make cDNA of both miRs and mRNA?

They claim that it wil add an artificial polyA tail and therefore both miRNA and mRNA can be reverse transcribed. This way you should be able to run qRT-PCRs for both miRs and mRNA.

Another advantage may be that you can use mRNA as a housekeeping gene

So far we've been using the AB Taqman miRNA RT kit, which is based on stem-loop RT for specifically miRs.

Thanks!
-mcb

[mirno1]

It should be possible (the artificial poly-a-tail+universal sequence will be added to all molecules if i understood it right). But using universal primer of qiagen for taqman realtime means a big amount of unnecessary costs. it´s much cheaper to use common sybr green and poly-t-primer for mRNA-RT, even if they are reverse transcribed together.
i wouldn´t use mRNA housekeeping genes as a control, as you don´t know if isolation, reverse transcription or amplification is favourable to bigger or smaller molecules.
However, i´d be interested what others think.

[miRNA_diabetes]

I think any of the housekeeping gene's mRNA could be used as a normalizer. I used the U6 small RNA as a normalizer for my qPCR results. But then again, I do qPCR from Total RNA.. no enrichment of miRNAs.

I hope this helps!!  

Edited by miRNA_diabetes, 23 October 2009 - 12:31 PM.

[Functional Screens]

miRNA_diabetes, on Oct 23 2009, 10:48 AM, said:

I think any of the housekeeping gene's mRNA could be used as a normalizer. I used the U6 small RNA as a normalizer for my qPCR results. But then again, I do qPCR from Total RNA.. no enrichment of miRNAs.

I hope this helps!!  

I am sorry, I do not agree.  In general, you might want to use the same method, e.g. adding polyA to the small RNA, to detect both miRNA and endogenous control.

[it_never_ends]

If I understand their (Qiagen's) claim correctly, they use an oligo-dT primer to amplify the polyA tailed mature-miR. Thats 'almost' the same procedure when we use oligo-dT primers for mRNA reverse-transcription.
If you don't know of a good miR to normalize for your system I'd say housekeeping mRNA's would be a good precursor atleast.

I have use the cDNA generated using the miSCript kit, and it works fine, for miRNA and mRNA qPCR - Disclaimer: I have no affiliations with Qiagen