Miscript sybr green qpcr kit

Total cellular RNA was extracted by TRIzol method (Invitrogen, Carsbad, California, USA) following the manufacturer’s instructions. After RNA extraction, an amount of 0.25 μg of total RNA was ready to be reversely transcribed to cDNA by using the miScript II RT Kit (Qiagen Tec, Shanghai, China). Then, we performed miScript SYBR Green qPCR Kit to complete the qPCR reaction on the Applied Biosystems 7500 FAST real-time PCR System (Applied Biosystems, Foster California, USA). Specific primers and probes for mature miR-205 and small nuclear RNA RNU6B (endogenous reference) were purchased from Sangon Biotech (Shanghai, China). All reactions were detected in triplicate. The amount of miR-205 was obtained by normalizing to small nuclear RNA RNU6B as control. The primers for detecting HMGB1, RAGE and GAPDH were as follows: 5′-CATCTCAGGGCCAAACCGAT-3′ (forward) and 5′-TGACATTTTGCCTCTCGGCT-3′ (reverse) for human HMGB1; 5′-GCTTGGAAGGTCCTGTCTCC-3′ (forward) and 5′-CCTCTGACACACATGTCCCC-3′ (reverse) for human RAGE; 5′-TGCACCACCAACTGCTTAGC-3′ (forward) and 5′-GGCATGGACTGTGGTCATGAG-3′ (reverse) for human GAPDH. The relative fold changes of candidate genes were analyzed by using method.