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.
You can use the EPIK miRNA kit from Bioline that is designed for using SYBR green qPCR. I did successfully for miRNA 146a-5p.
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