Total RNA were extracted with TRIzol reagent, and miRNAs were extracted by TRIzol SM Reagent (HaiGene, Haerbing, China). After quantified by spectrophotometer, equal RNA of each sample was reversely transcribed into cDNA using PrimeScript® miRNA or mRNA cDNA synthesis kit (TaKaRa, Tokyo, Japan). The generated cDNA was amplified using Platinum SYBR Green qPCR Super Mix UDC (Invitrogen, Carlsbad, CA) in an ABI Prism 7000 (Applied Biosystems, Foster City, CA). The genes encoding mouse MEG3, 12/15-LOX mRNA, miR-181b were amplified with the following primer pairs. Relative expression of these genes was calculated by the 2−ΔΔCT value method. Relative expression of MEG3 and 12/15-LOX mRNA was normalized to GAPDH, and relative expression of miR-181b was normalized to U6.
Primers for MEG3, 12/15-LOX mRNA, miR-181b are as follows:
Primers for MEG3, 12/15-LOX mRNA, miR-181b are as follows:
MEG3: 5′-CTGCCCATCTACACCTCACG-3′ (sense) and 5′-CTCTCCGCCGTCTGCG CTAGGGGCT-3′ (antisense)
12/15-LOX: 5′-ACCCCACCGCCGATTTT-3′ (sense) and 5′- AGCTTCGGACCCAGCATTT-3′ (antisense)
miR-181b: 5′-CAGACATCTCTGCCTCACA-3′(sense) and 5′-TTGCGGTTCTGTCTTCAGC-3′ (antisense)