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[veer2]

Ask for help about the miRNA qRT-PCR by SYBR green methods
I just start to work on the miRNA qRT-PCR fields and try to establish some assay, the method is firstly to add poly (A) at the 3-end of miRNA, then use SYBR green to perform qPCR, I am still confused about some points:
1. After the isolation of total RNA (Trizol methods from invitrogen), add poly (A) at the 3-end of  miRNA,  then use oligo d(T)n to synthesis the first cDNA strand:
How many Ts should be added? At least 15 Ts should in the oligo d(T)15 or even more??

2. From the publications, at the 3-end of oligo d(T)n, 2 random base V (A, G, C) and N (A, G, C,T) should be added for the anchor function, Are these 2 bases V/N totally randomly? If so, in the new synthesised first strand cDNA strain, there will be 2 base mismatch, will it affect the  further qPCR experiments?? Or the V/N must be the complementary base of the 3-end of the mature miRNA which are expected to be tested?

3. at the 5-end of the oligo d(T)n, there should be some universal adaptor, This adaptor should include the universal sequence for the further qPCR. (the sequence should be used as the reverse primer further). Is the sequence of the universal adaptor totally randomly besides including the primer sequence? Or Any standard and requirements for the sequence of the universal adaptor and universal PCR primer?

4. After poly (A) is added, the solution is directly for the further first cDNA reverse transcript, or it should be purified by phenol-chlororm methods?

Thanks a lot in advance,
veer