Primerscript rt reagent kit with a gdna eraser

Total RNA was extracted from tissues and cells with TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The miRNAs were extracted from tissues with RNAiso for Small RNA reagent (Takara, Tokyo, Japan). Total RNAs were reverse-transcribed into complementary cDNA using a PrimerScript™ RT reagent kit with a gDNA eraser (Takara, Tokyo, Japan) according to the manufacturer's protocols. The PCR procedure was as follows: one cycle at 95°C for 30 seconds, followed by 40 cycles of 95°C for 5 sec and 60°C for 31 seconds. Melting curve analysis was performed at 65~ 95°C. The miRNAs were polyadenylated and subsequently converted into cDNAs using a Mir-X™ miRNA First-Strand Synthesis kit (Clontech Laboratories, Mountain View, CA, USA) according to the manufacturer's protocols. The PCR procedure was identical to that mentioned above. All the data obtained were calculated by the 2^-ΔΔCt method. We used GAPDH as an internal reference for HOTAIR and Cx43. U6 was used as the internal standard for normalizing gene expression of miR-613. All primer sequences for RT-qPCR are listed in Table 2.

Twelve differentially expressed genes were selected for qRT-PCR analysis (Table S5). The Oligo7 (version 7.56) software was used to design primers. PrimerScript RT reagent kit with a gDNA eraser (Perfect Real Time) (TaKaRa, RR047A) was used to synthesize cDNA according to the manufacturer’s protocol. Three technical replicates were performed for each sample. The final reaction volume of 10 µL contained 2 µL of cDNA, 8 µL of qPCR master mix (5 µL of TB Green Premix Ex Taq II system according to the manufacturer’s instructions (TaKaRa, RR820A), 0.25 µL of each primer, and 2.75 µL of RNA-free water). The samples were run on a CFX96 Touch Real-Time System (Bio-Rad, Hercules, CA, USA). The conditions were 94 °C for 30 s, followed by 39 cycles of 94 °C for 5 s and 65 °C for 34 s. The relative mRNA levels of the candidate key genes were calculated using the 2^–ΔΔCt method.