Superscript 3 first strand synthesis supermix

Total RNAs in passage 4 BM-MSCs were extracted using Trizol Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. RNA integrity was determined using formaldehyde denaturalization agarose gel electrophoresis. RNA concentrations were measured by the smartspec^TM plus spectrophotometer (BIO-RAD, Hercules, CA, USA). cDNA was generated using SuperScript III First Strand Synthesis SuperMix (Takara, Dalian, China) or a TaqMan MicroRNA Reverse Transcript Kit (Applied Biosystems). TaqMan probes for individual miRNAs were purchased from Applied Biosystems. β-Actin and U6 was used as internal housekeeping controls, respectively. Quantification of mRNA and mature miRNA was performed on an ABI 7500 real-time PCR detection system (Applied Biosystems, USA) as previously described. The specific primers oligonucleotides (TaKaRa, Dalian, China) were used and relative expression of the target genes was calculated with the 2^−ΔΔCt method.

cDNA was synthesized from Trizol-isolated total RNA by use of the SuperScript III First Strand Synthesis SuperMix for quantitative reverse transcribed polymerase chain reaction (qRT-PCR; Takara) according to the manufacturer’s instructions. For real time PCR experiments, reactions containing the SYBR Premix EX Taq^TM (Takara), ROX Reference Dye (50 × , Takara), cDNA and gene primers were run on the StepOnePlus^TM Real Time PCR Systems and analyzed with StepOne Softwase V2.1 (Applied Biosystems). Relative quantification was calculated using the comparative Ct method45 (link). The primers for different genes were listed in Table 2.