Sybr select master mix kit

According to the standard protocol, the total cell RNA solution was extracted with Trizol (Invitrogen, Carlsbad, United States). RNA concentration was validated by Nanodrop (Invitrogen, United States), followed by in vitro cDNA synthesis with PrimeScriptTM RT Master Mix kits (Takara, Japan). Then SYBR^® Select Master Mix kit (designed for total RNA) was used for RT-qPCR analysis on the Bio‐Rad CFX96 qPCR instrument (Bio‐Rad, Hercules, CA). β-Actin was selected as a reference gene for the coding mRNA test, while U6 was used as a reference gene for the microRNA test. The primers were: PTEN-forward:5′-CAA GAT GAT GTT TGA AAC TAT TCC AAT G-3′; PTEN-Reverse: 5′-CCT TTA GCT GGC AGA CCA CAA-3′; miR-21-5′-GCC CGC TAG CTT ATC AGA CTG ATG-3′; β-actin-F-5′-CTG AAC CCT AAG GCC AAC CG-3′; β-actin-R-5′-GTC ACG CAC GAT TTC CCT CTC-3′; U6-F-5′-GCT TCG GCA GCA CAT ATA CTA AAA T-3′; U6-R-5′-CGC TTC ACG AAT TTG CGT GTC AT-3′.

Trizol purchased from Invitrogen (Calsbad, USA) was used to extract total cell RNA as standard protocol. RNA concentration was tested by Nanodrop (Invitrogen, USA). PrimeScriptTM RT Master Mix kits were used to synthesis cDNA; and Taqman MirNA assay kit (designed for microRNA) and SYBR® Select Master Mix kit (designed for total RNA) were used for RT-qPCR analysis on Bio-Rad CFX96 qPCR instrument (Bio-Rad,Hercules, CA). All these kits were purchased from Takara (Japan). β-actin and U6 were used as reference genes.