Miscript pcr kit

Total RNA was isolated from tissue samples or cultured cells using Qiazol reagent (Qiagen, Gaithersburg, MD, USA) or TRIzol reagent (Invitrogen, Carlsbad, CA, USA). RNA was reverse‐transcribed into miRNA cDNA or total cDNA using the Prime Script miRNA cDNA synthesis kit (Qiagen) or PrimeScript RT kit (Qiagen), and then subjected to quantitative RT‐PCR (qRT‐PCR). mRNA or miRNA expression was determined by qPCR on a Q6 real‐time PCR System (Applied Biosystems, Carlsbad, CA, USA) using SYBR Green Master Mix (4309155, Applied Biosystems) or the miScript PCR Kit (Qiagen), using GAPDH or U6, respectively, as the internal loading control. Reverse transcription and PCR were performed as described in our previous report.⁵⁵ Specific primers for human MMP9 (Forward: 5′‐TTC CAA ACC TTT GAG GGC GA‐3′ and Reverse: 5′‐CTG TAC ACG CGA GTG AAG GT‐3′), human SMURF1 (Forward: 5′‐AAC TGA AAC CCA ATG GCA GAA ATGT‐3′ and Reverse: 5′‐TTG CCA GAA CCA CCG CAC GAT G‐3′), and human GAPDH (Forward: 5′‐GAC TCA TGA CCA CAG TCC ATG C‐3′, Reverse: 5′‐CAG GTC AGG TCC ACC ACC ACT GA‐3′) were used for PCR amplification. The primer for miR‐516a (5′‐TGC TTC CTT TCA GAG GGT‐3′) was synthesized by Sunny Biotechnology (Shanghai, China), and U6 was used as the internal loading control through the primer provided in the miScript PCR Kit. The data were analyzed as previously described.⁵⁵

Total RNA was reverse transcribed using the miScript Reverse Transcription Kit (Qiagen, Valencia, CA). RT-qPCR was performed using the miScript PCR Kit (Qiagen). Experiments were normalized to U6. Results were reported as RQ with respect to a calibrator sample using the 2-ΔΔCt method. MiR-200a, miR-200b, miR-200c and U6 kits were purchased from Biomics biotech (NanTong).