Sequence detection software v1.4 7300 real time pcr system

Total RNA in the cells was extracted using Trizol reagent (Invitrogen, Thermo Fisher Scientific, Rockford, IL, USA), according to the manufacturer’s instruction. Five-hundred nanogram of total RNA was reversely transcribed using a reverse transcription kit (TaKaRa Biotechnology Co., Ltd., Dalian, Liaoning, China). The primer sequences of human B2M were 5′-GGTTTCATCCATCCGACATTG-3′ (forward) and 5′-CATGTCTCGATCCCACTTAAC-3′ (reverse). The primer sequences of human β-actin were 5′-ACAATGTGGCCGAGGACTTT-3′ (forward) and 5′-GCACGAAGGCTCATCATTCA-3′ (reverse) (synthesized by Sangon Biotech Co., Ltd., Shanghai, China). PCR amplification was performed at 95 °C for 5 s and 60 °C for 30 s for 40 cycles using a SYBR Premix TaqTM II (Tli RNaseH Plus) kit (TaKaRa Biotechnology Co., Ltd.) with an initial step of denaturing RNA at 95 °C for 30 s. Assays were conducted in triplicate and repeated at least three times. The amount of target (B2M) normalized to an endogenous control (β-actin) given by 2^ΔΔCt, in which threshold cycle (Ct) was obtained using Sequence Detection Software v1.4 (7300 Real Time PCR System, Applied Biosystems, Foster City, CA, USA).

Total RNA was extracted using RNAiso Plus (Takara, Japan) according to the manufacturer's instructions. Reverse transcription was performed using a PrimeScript™ RT Master Mix (Takara, Japan) with specific primers (see Table 1). Reaction conditions were followed as 37°C 15 min, 85°C 5 s, 4°C 30 min. PCR amplification was performed at 95°C 10 min, followed by 95°C 5 s and 60°C 31 s for 40 cycles using SYBR Green Master. Assays were conducted in triplicate and were repeated at least three times. The amount of DHCR24 normalized to an endogenous control GAPDH given by 2-ΔΔCt, in which threshold cycle (Ct) was obtained using Sequence Detection Software V1.4 (7,300 Real Time PCR System, Applied Biosystems, USA).