High capacity cdna reverse transcription kit cdna kit

Briefly, total RNA was extracted using Trizol (Invitrogen; Thermo Fisher Scientific, Waltham, MA, USA), and for evaluating the RNA quality, the A260/A280 ratio was analyzed using the NanoDrop VR ND-1000 Spectrophotometer (NanoDrop Technologies; Wilmington, DE, USA). For cDNA synthesis, a High-Capacity cDNA Reverse Transcription Kit cDNA Kit (Applied Biosystems™, USA) was used, followed by the preparation of the primers according to their manufacturer instructions, Sangon Biotech (Beijing, China), as provided in Table 1.
Real-time RT-PCR was performed in Mx3005P real-time PCR system (Agilent Stratagene, USA) using TOPrealTM qPCR 2X PreMIX (SYBR Green with low ROX) (Cat. # P725 or P750) (Enzynomics, Korea) following the manufacturer’s instructions. The PCR cycling conditions included initial denaturation at 95 °C for 12 min followed by 40 cycles of denaturation at 95 °C for 20 s, annealing at 60 °C for 30 s, and extension at 72 °C for 30 s. A melting curve analysis was performed following PCR amplification. The expression level of the target genes was normalized using the mRNA expression of a known housekeeping gene, Gapdh. Results are expressed as fold-changes compared to the control group following the 2^−ΔΔCt method [100 (link)].