Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) detection

A549 and HCT116 cells were treated with SAHA (0.5 µM) or TSA (100 nM) at 37°C for 24 h. Treatment with DMSO (equal volume added) was used as the control. Total mRNA was extracted from A549 and HCT116 cells using TRIzol reagent. RNA sample concentration was measured using a UV spectrophotometer, and optical density (OD)260/OD280 was limited to 1.8–2.0. A total of 500 ng RNA was used for cDNA synthesis with the PrimeScript^® RT reagent kit. Subsequently, qPCR was performed on an ABI 7500 Real-Time PCR system (Thermo Fisher Scientific, Inc.) using the Takara SYBR^® Premix Ex Taq™ kit to quantify the expression of target genes. Primers used in qPCR experiments are presented in Table I, with GAPDH serving as an internal reference. The thermal cycling conditions for qPCR were as follows: Holding stage conducted at 95°C for 30 sec; cycling stage conducted at 95°C for 5 sec and 60°C for 34 sec for 40 cycles; melt curve stage conducted at 95°C for 15 sec, 60°C for 60 sec, 95°C for 30 sec and 60°C for 15 sec. Following normalization to the GAPDH gene, the expression of each target gene was calculated using the comparative cycle quantification (Cq) method (24). In correlation analysis, the ΔCq values were calculated according to the following formula: ΔCq=Cq (gene of interest)-Cq (GAPDH). For determination of the relative expression, the 2^−ΔΔCq value was calculated according to the following formula: ΔΔCq=ΔCq (control group)-ΔCq (experimental group).

Primers used in reverse transcription-quantitative polymerase chain reaction assay.

ABC, ATP-binding cassette.