miRNA real time PCR by SYBR green methods - (Jun/08/2009 )

I am working on miRNA real time PCR detection. The strategy is: 1. add poly A tails at the end of miRNA 2. add oligo d(T)12 adaptor to synthesis the first strand cDNA. 3. real time PCR to test mature miRNA

I am confused about some points:
1. the adaptor for the first strand cDNA is: oligo d(T)n VN to anchor the beginning site of the 3 end of the miRNA, if so, if the 3 end of the miRNA is base-A or AA, it means the adaptor VN will start to anchor the last NOT A base pair in the miRNA and the first strand cDNA will lose two T bases pairs. Is it right?

2. How to distinuish the miRNA family which could one or two base pair mismatc? for example, let-7a, let-7b, let7C.... by real time PCR? change the anealing temperature?

3. what is the difference for the two steps real time PCR program and three steps real time PCR program? two steps (anealing and extension at 60 C together;) three steps ( anealling at 60C and extension at 72C). Is it three steps real time PCR will provide a better specificity?

Thanks in advance,

veers

This won't answer any of your questions (sorry!), but why are you developing such an assay? There are very nice TaqMan assays for (I think) all miRNAs in mirbase, and if these are too expensive there are also sybr green methods previously published (although in my experience, at least the Ambion sybr method, is not very good). So I suppose my question is what advantage will your system have over the ones already developed? If you have a novel miRNA for which there are no primer sets, I believe that ABI will design a custom miRNA TaqMan assay for you.