Hairpin ittm mirnas qpcr quantitation assay kit

Total RNAs were extracted using the TRIzol agent (Ambion, ThermoFisher Scientific, Waltham, MA, USA) according to the instruction of the manufacturer. The reverse transcription of RNA and quantitative real-time PCR was performed using the Hairpin-it^TM miRNAs qPCR Quantitation Assay Kit (GenePharma, Shanghai, China) according to the manufacturer’s instructions. Quantitative RT-PCR was performed using a Roche 480 real-time PCR system. The 2^−ΔΔCt method was used to evaluate miR-654-5p gene expression after normalization for expression of the endogenous controls U6 (U6 non-coding small nuclear RNA). All primers for miR-654-5p and the U6 genes were designed, synthesized, and verified using GenePharma. Mature miR-654-5p sequence was as follows: 5′-UGGUGGGCCGCAGAACAUGUGC-3′. Each experiment was repeated at least three times.

Total RNAs were extracted using the TRIzol agent (Ambion) according to the instruction of the manufacturer. Reverse transcription of RNA and quantitative real-time PCR was performed using the Hairpin-it^TM miRNAs qPCR Quantitation Assay Kit (GenePharma) according to the manufacturer's instructions. Quantitative RT-PCR was performed in a Roche 480 real-time PCR system. The 2^−ΔΔCt method was used to evaluate the miR-622 gene expression after normalization for expression of the endogenous controls U6 (U6 non-coding small nuclear RNA). All primers for miR-622 and the U6 genes were synthesized and approved by GenePharma. Each experiment was repeated at least three times.