Sybr green pcr master mix

Total RNA was extracted using an RNA kit and protocol (GenePharma Co., Ltd., Shanghai, China). mRNA contained in 2 µg total RNA was reverse transcribed using a Transcriptor strand cDNA synthesis kit (GenePharma Co., Ltd.). PCR amplification was performed using a SYBRGreen PCR Master Mix (GenePharma Co., Ltd.) and the primers listed subsequently (Integrated DNA(deoxyribonucleic acid) Technologies [IDT], 300 nM). mRNA abundance was quantified using the threshold cycle method.
Collagen I: 5′-TGCTGCCTTTTCTCTCCTT-3′, 5′-AAGTGCTGGTAGGGAAT-3′;
Runx2: 5′-TGCTGGAGTGATGTGGTTTTCT-3′, 5′-CCCCTGTTGTGTTGTTTGGTAA-3′;
Aggrccan: 5′-GCATCCGAAACCCTGTAAC-3′, 5′-GGCGGTCAGCATCATAGTCC-3′;
OPN: 5′-CAAATACCCAGATGCTGTGGC-3′, 5′-TCCTGGCTGTCCACATGGAC-3′;
IRKA4: 5′-ACTTCTTGTACGAGGTGCCGCC-3′, 5′-GGGCAGGCCTGGGTCTGGCAGT-3′;
IKKα: 5′-CCGCTCGAGATGGACCGTTGCTACGATCC-3′, 5′-GGGGTACCTCAGTGCACCTGAGGCTG-3′;
IKKβ: 5′-GCCAGAAAACATCGTCCT-3′, 5′-CACCGTTCCATTCAAGTC-3′; and
NF-κB: 5′-GGAGGCATGTTCGGTAGTGG-3′, 5′-CCCTGCGTTGGATTTCGTG-3′.

RNA was extracted from cells or tissues using TRIzol (Invitrogen). miR-181b (qRT-PCR) reactions were performed using Fermentas reverse transcription reagents and SYBR Green PCR Master Mix (GenePharma) according to manufacturer's protocols. U6 was used for normalisation. Relative gene expression was calculated by the 2^−∆∆Ct method.