Rnazol rt rna isolation reagent

Total RNA was extracted from plasma using RNAzol® RT RNA Isolation Reagent (Sigma-Aldrich). High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific) was used to performed reverse transcription. To detect the expression of lncRNA TUG1 and PTEN mRNA, Luna® Universal One-Step RT-qPCR Kit (NEB) was used to prepare PCR reaction systems. Primers of lncRNA TUG1 and β-actin were designed and synthesized by GenePharma (Shanghai, China). The expression of lncRNA TUG1 was normalized to endogenous controls β-actin using the 2^−ΔΔCT method.

Tissue specimens were ground in liquid nitrogen, followed by the addition of RNAzol^® RT RNA Isolation Reagent (Sigma-Aldrich). In vitro cultured cells were directly mixed with RNAzol^® RT RNA Isolation Reagent to extract total RNA. Expression levels of POU3F3 and MEG3 were measured by RT-qPCR. Briefly, total RNA samples were reversely transcribed into cDNA using SuperScript IV Reverse Transcriptase (Thermo Fisher Scientific). Luna^® Universal One-Step RT-qPCR Kit (NEB) was used to prepare PCR mixtures with SYBR Green (NEB) as the fluorophore. With GAPDH as endogenous control, qPCRs were performed through the following conditions: 2 min at 95 °C, followed by 40 cycles of 15 s at 95 °C and 40 s at 55 °C. All data normalizations were performed according to the 2^−ΔΔCT method [18 (link)]. Primer sequences were: MEG3 forward 5′-CTGCCCATCTACACCTCAC-3′ and reverse 5′-CTCTCCGCCGTCTGCGCTA GGG-3′, POU3F3 forward 5′-TCATCCTTCAGRGRCCATCC-3′ and reverse 5′-ATC TCAGATTCCTGGGCTGG-3′, GAPDH forward 5′-ACCACAGTCCATGCCATCA-3′ and reverse 5′-CCACCACCCTGTTGCTGTA-3′.

To detect PTCSC3 expression, total RNAs were extracted using RNAzol® RT RNA Isolation Reagent (Sigma-Aldrich, St. Louis, MO, USA) and reverse transcribed into cDNAs using RevertAid RT Reverse Transcription Kit (Thermo Fisher Scientific). All PCR reactions were prepared using SYBR® Green Quantitative RT-qPCR Kit (Sigma-Aldrich, St. Louis, MO, USA) and carried out on Applied Biosystems 2720 Thermal Cycler with GAPDH as the endogenous control. Primers of PTCSC3 and GAPDH were designed and synthesized by Sangon (Shanghai, China). PTCSC3 expression levels were normalized to GAPDH using the 2^−ΔΔCt method.