Sybr premix ex taq master mix

Quantitative RT-PCR was carried out using SYBR Premix Ex Taq Master Mix and a 2-Step kit (TaKaRa, Dalian, China). Total cellular RNA was isolated using TRIzol reagent according to the manufacturer’s instructions (Life Technologies). Total RNA (2.0 μg) was used for cDNA synthesis using oligo dT primers (Transgene, Beijing, China), and 1/10 of the cDNA volume was used for quantitative PCR. PCR amplification was carried out using a CFX96 Touch System (BioRad, Hercules, CA, USA). The PCR conditions were carried out according to the manufacturer’s instructions. The Ct values were normalized to that of the human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene. The ΔΔCt method was used to determine the relative expression level of the target genes. All samples were run in triplicate in each experiment. Each assay was repeated three times. All primers were synthesized by TSINGKE (Beijing, China). The primers were as follows: for HCF-1, 5’ - GGC AGT GCT CTG ATT TCC A -3’ and 5’ - TTC AGG ATT GTT CCC GCT-3’; for CDC42, 5’ - GGA TTA TGA CAG ATT ACG ACC G-3' and 5’ - GTT ATC TCA GGC ACC CAC TTT-3’; and for GAPDH, 5’ - AAA TTG AGC CCG CAG CCT-3' and 5’ - CCC AAT ACG ACC AAA TCC GT-3’.

Total RNA in tissues and cells was isolated with TRIzol (Invitrogen). Two hundred nanograms of total RNA was used to synthesize the first-strand cDNA with the Reverse transcription kit (Abcam, Cambrige, UK). The quantitative PCR was performed with SYBR Premix Ex Taq Master Mix (TaKaRa, Shiga, Japan) on ABI 7900 real-time PCR system (Promega). GAPDH was used as an internal control to LINC00461 and AQP4 mRNA, and U6 small nuclear RNA (U6) was for mature miR-216a. The relative expression was calculated according to the comparative threshold cycle value (2^−ΔΔCt) method. The reactions were performed in triplicate for each sample at least three independent runs, and the primers involved are as follows: LINC00461, 5′-GACATTTACGCCACAACCCACG-3′ (sense) and 5′-AGACAGACCCTCAGATTCCCCA-3′ (anti-sense); miR-216a, 5′-ATCCAGTGCGTGTCGTG-3′ (sense) and 5′-TGCTTAATCTCAGCTGGCA-3′ (anti-sense); AQP4, 5′-GTGACAGACCCACAGCAAGG-3′ (sense) and 5′-TCAACTCAACCAAGGAGACCAT-3′ (anti-sense); GAPDH, 5′-AGGTCGGAGTCAACGGATTT-3′ (sense) and 5′-ATCTCGCTCCTGGAAGATGG-3′ (anti-sense); and U6, 5′-GCTTCGGCAGCACATATACTAAAAT-3′ (sense) and 5′-CGCTTCACGAATTTGCGTGTCAT-3′ (anti-sense).