Mirna specific assay kit

Total RNA, including small RNAs and mRNA, was extracted using the TRIzol reagent (Invitrogen, USA) according to the manufacturer’s instruction. The concentration of RNA was determined using a NanoDrop spectrophotometer (NanoDrop, USA). The miR-497 stem-loop primer, U6 primer, mRNA primer, and GAPDH primer were bought from Guangzhou RiboBio Co., LTD. The expression of miR-497 was assayed using stem-loop RT, followed by real-time PCR analysis. MiR-497 cDNA was synthesized from total RNA using the miRNA reverse transcription kit (TaKaRa, Dalian, China), and the expression levels of miR-497 were quantified using the miRNA-specific assay kit (TaKaRa, Dalian, China). U6 snRNA was used as an internal control. Reverse transcription of the mRNAs were performed using the PrimeScript RT reagent kit (TaKaRa, Dalian, China), and the expression levels of the mRNAs were determined using SYBR Premix Ex Taq (TaKaRa, Dalian, China), with GAPDH as an internal control. Real-time PCR was performed on a real-time PCR instrument (CFX96, BIORAD, USA). The results of the qRT-PCR analysis were determined based on the threshold cycle (Ct), and the relative expression levels were calculated using the 2^-ΔΔCt method, after normalization to the expression of the internal control gene.

Expression levels of miR-148b were determined using the miRNA-specific assay kit (Takara, China), and U6 was used as an internal control. Expression levels of Bcl-w were detected using SYBR Premix Ex Taq II (Takara, China), with β-actin as an internal control. Quantitative real-time PCR (qRT-PCR) were performed on an ABI PRISM® 7500 Sequence Detection System. The signals were normalized to the internal control and expressed as 2^-ΔΔCt.