I am going to detect miR-146a in human cell lines by RT PCR. I found most of the people are using the taqman assay kit. In my lab superscript III SYBR green method is already established and we are having a light cycler 2.0. Can I go for SYBR green method for detection of miR-146a? Can you suggest a complete protocol? Thanking you.
You can use miScript assays from Qiagen.
http://www.qiagen.com/de/products/catalog/assay-technologies/mirna/miscript-primer-assays/
Indeed, miScript works very well. To isolation I recommend
miRNeasy Mini Kit from Qiagen as well.
inspite of using kit can I use custom designed primers for mature mirna?
I dont see how you can amplify mature miRNA using traditional PCR method. The mature miRNAs are too short for 2 primers to bind for a pcr reaction. However you can use the mature miRNA as if it is one of the primers and use a template that has specific site for the miRNA and a second primer specific to the other end.
Also you might want to check out the "gold standard" in mature miRNA detection: northern blotting.
Dear Pooladanda
In our lab to detect mature miRNA at first we add a poly A tail to all transcripts by polyA polymerase, then we use a synthetic adaptor which has several T in its 5'. Using a primer specific to the terminal sequence of the adaptor we make cDNA.To perform SYBR Green qPCR in addition to mentioned primer, we add a primer specific to a given mature miRNA.
I hope this will be helpful for you
synthetic adoptor T means RT primer?
Thank youn sir, could you please forward that protocol to me.
Sir, I think using an RT-primer specific to miR-146 and superscript lll (Invitrogen) for cDNA synthesis is a very good option. You make look for the protocol in Gasic et al, Plant methods (2007).
Hope it will help.
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