Moloney murine leukemia virus rt kit

Total RNA was extracted from the cultured cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Total RNA was reverse transcribed into cDNA using the Moloney murine leukemia virus RT kit, with the M-MLV buffer, dNTP and random primers (all from Promega Corporation). The temperature protocol for the reverse transcription reaction consisted of cDNA synthesis at 37˚C for 60 min and termination at 80˚C for 2 min. qPCR was subsequently performed using the SYBR Green Realtime PCR Master Mix (Beijing Solarbio Science & Technology Co., Ltd.) in a Bio-Rad CFX96 system (Bio-Rad Laboratories, Inc.), according to the manufacturer's protocols. The primer sequences used for qPCR were designed and synthesized by Guangzhou RiboBio Co., Ltd. (Table I). The following thermocycling conditions were used for the qPCR: Initial denaturation at 95˚C for 2 min, followed by 40 cycles 94˚C for 20 sec and 60˚C for 30 sec, and final extension at 72˚C for 30 sec. Relative mRNA or miRNA expression levels were calculated using the 2^-DDCq method (33 (link)) and normalized to the internal reference genes GAPDH and U6, respectively. All experiments were performed in triplicate.

Cellular RNA was isolated using TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Subsequently, DNA was removed from the samples via DNase treatment (DNA-free kit; Ambion; Thermo Fisher Scientific, Inc.) and cDNA was synthesized from the purified RNA using a Moloney murine leukemia virus RT kit (Promega Corporation, Madison, WI, USA). GAPDH primer sets were used as a normalization control. Primer sequences were as follows: GAT1 forward, 5′-GCAATCGCCGTGAACTCTTC-3′ and reverse, 5′-AGGAAATGGAGACACACTCAAAGA-3′; EAAC1 forward, 5′-CTCCACCACCGTCATTGCT-3′ and reverse, 5′-TGGCAGGCTTCACTTCTTCAC-3′; GAPDH forward, 5′-GTATGTCGTGGAGTCTACTG-3′ and reverse, 5′-CTTGAGGGAGTTGTCATATTTC-3′. RT-qPCR cycling conditions were: Initial denaturation for 3 min at 95°C followed by 45 cycles of 95°C (10 sec) and 58°C (45 sec), and data were acquired at the end of the annealing/extension phase. RT-qPCR was performed in triplicate using the SYBR-Green PCR Master Mix (Applied Biosystems) on a 7900HT Fast Real-Time PCR machine according to the manufacturer's protocol (Applied Biosystems; Thermo Fisher Scientific, Inc.). The relative quantification method (2^−ΔΔCq) (19 (link)) was used to analyze quantitative RT-PCR data using GAPDH as normalizer. NC were served as a reference.