I have some question about miRNAs. After making in silico analyze via TargetScan or the other web tools, I see 8mer, 7mer-m8. Which is the best matching? Also I see poorly conserved and highly conserved writings. Should I select the miRNA with highly conserved? or if I choose poorly conserved miRNA, is it false in theory?
After choosing miRNA, I want to do some functional assays such as qRT-PCR with miRNA\’ s target mRNA. In example hsa-miR-145 match with TPM3 (NM_001043351, tropomyosin 3) gene. When I design primers and prob for this gene, should I consider the miRNA macthing site? Should the primers and probe localized in miRNA:mRNA site?
Thanks for your attention