Real-time PCR Quantification of miRNA and mRNA

Total RNA from the frozen tissues or cultured cells was isolated with mirVana^TM PARIS^TM Kit (Ambion, USA) according to the manufacturer’s instructions. RNA was first reversely transcribed into cDNA by using RT reagent Kit (TOYOBO, Japan). Then the cDNA was subjected to real-time PCR with an SYBR^Ⓡ Green Realtime PCR Master Mix kit (TOYOBO, Japan) in an ABI PRISM 7500 system (Applied Biosystems, USA) by using the miR-29a primers set and U6 primers set (Ribobio, China). Human VEGF-A primers for RT-PCR were produced by the Sangon Inc., Shanghai, China. The sequence of VEGFA reverse primer is (34) (link) : 5’-ATGATTCTGCCCTCCTCCTT-3’; and the forward primer is: 5’-CCTTGCTGCTCTACC TCCAC-3’ (74 bp). The relative quantification of RNA expression was calculated using the 2^-ΔΔCt method (35) (link).