Primescript reverse transcription reagent kit with genomic dna eraser

TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) was used to isolate RNA from Y79 cells. Subsequently, cDNA was synthesized from RNA using the PrimeScript™ reverse transcription (RT) reagent kit with genomic DNA Eraser (Takara Biotechnology Co., Ltd., Dalian, China) according to the manufacturer's protocol. The forward (5′-CCCAAGCTTATGCGTTCCCCACGGACGC-3′) and reverse (5′-CCGGAATTCCTATACGATGTACTCCATTCGGTTTAAGCTC-3′) primers were designed for cloning the coding sequence of JAG1 using the cDNA extracted from Y79 cells as a template. The polymerase chain reaction (PCR) was performed using the PrimeSTAR HS DNA polymerase (Takara Biotechnology Co., Ltd.) with the conditions as follows: Initial denaturation at 94°C for 10 min followed by 30 cycles each consisting of 98°C for 20 sec, 50°C for 20 sec, and 72°C for 5 min and a final extension at 72°C for 10 min. The obtained DNA was subsequently cloned into a pcDNA3.1(+) plasmid (Invitrogen; Thermo Fisher Scientific, Inc.). The generated recombinant plasmid pcDNA-JAG1 was sequenced by Sangon Biotech Co., Ltd. (Shanghai, China). Cells (2×10⁵/well) was transfected with 500 ng plasmid using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Following incubation for 48 h, cells were harvested for further experimentation