Ribozol reagent

Extraction of total RNA from tissues and cells was performed using Ribozol reagent (Invitrogen, USA). AMV Reverse Transcriptase XL (Clontech, USA) and QuantiTect SYBR Green PCR Kit (Qiagen, Shanghai, China) were used to prepare reverse transcription and qPCR reaction, respectively. With 18S rRNA as endogenous control, the expression level of PLAC2 was normalized. With GAPDH as endogenous control, PTEN expression level (a target of miR-21) was normalized.
To detect miRNA-21, mirVana miRNA Isolation Kit (Thermo Fisher Scientific), qScript microRNA cDNA Synthesis Kit (Quantabio, USA) and miScript SYBR Green PCR Kit (QIAGEN, Germany) were used to perform miRNA extractions, miRNA reverse transcriptions and qPCR reactions, respectively. U6 was used as the endogenous control of miRNA-21. Three replicates were set for each experiment, and 2^-ΔΔCT method was used to process data.

Total RNA was isolated from paired tissue samples and H2170 cells using Ribozol reagent (Invitrogen). After being treated with DNase I (Invitrogen) for 100 min at 37 °C to completely remove genomic DNAs, RNA integrity was examined by electrophoresis on 6% urea-PAGE gels. The purity of RNA samples was reflected by OD260/280 ratios.

UM-SCC-17A cells were harvested at 24 h after transfections and mixed with Ribozol reagent (Invitrogen, USA, 10⁵ cells with 1 ml solution) to extract total RNAs. Tissues were ground in liquid nitrogen, followed by the addition of Ribozol reagent (0.5 g tissue with 1 ml solution) to extract total RNAs. RNA samples were treated with DNase I to remove genomic DNA. RNA samples were then used as template to synthesize cDNA through reverse transcription (25 °C for 10 min, 55 °C for 20 min and 85 °C for 10 min) using AMV Reverse Transcriptase (Canvax Biotech, USA). SYBR Green Master Mix (Bio-Rad, USA) was used to prepare RT-qPCR mixtures. The expression of CASC15 and cyclin D1 were detected using 18S rRNA and GAPDH as endogenous control, respectively. Using the same amount of cells and tissues (0.5 ng tissues with 1 * 10^–6 ml solution), miRNA extractions were performed using miRNA Isolation Kit (Geneaid, USA). After that, reverse transcriptions and RT-qPCRs were performed using All-in-One™ miRNA RT-qPCR Detection Kit* (Genecopoeia, Guangzhou, Shanghai). The expression of miR-365 was determined with U6 as the endogenous control. Three replicates were included for each experiment and 2^−ΔΔCT method was used to process data.

Total RNAs were extracted from cells from each transfection group and paired tissues from each patient using Ribozol reagent (Invitrogen) and treated with DNase I (Sangon) to remove genomic DNAs until an OD 260/280 ratio close to 2.0 was reached. RNA integrity was analyzed by separating RNAs on 5% urea-PAGE gels.