Reverse transcription-polymerase chain reaction (RT-PCR)

TRIzol^® reagent (Leagene, Beijing, China) was used to extract total RNA from the treated and untreated cells. The extracted total RNA was then reverse transcribed. Briefly, 2 µg of total RNA was added to the pre-existing mixture of 1 µl 10X reaction buffer (Leagene) with MgCl₂ and 1 µl DNase I. The non-specific inhibitor diethylpyrocarbonate (DEPC)-treated water was added to increase the volume to 10 µl, followed by incubation for 30 min at 37°C. Subsequently, 1 µl Oligo dT18 was added and mixed gently. A centrifugal separation step at 1,000 × g was performed after 5 min incubation at 70°C. The tube was kept on ice. For reverse transcription, 5 µl 5X Moloney Murine Leukemia Virus (M-MLV) buffer, 1.25 µl deoxyribonucleotide (dNTP) mixture, 1 µl M-MLV, 0.5 µl RNasin and DEPC-treated water were added until a total volume of 25 µl was achieved. Incubation was performed again for 15 min at 72°C. RT-PCR was performed in a total volume of 25 µl and the constituents used were: 2.5 µl 10X PCR buffer, 0.5 µl dNTP mixture, 0.625 µl MBI TaqDNA polymerase, 1 µl primer 1 (10 µmol/l), 1 µl primer 2 (10 µmol/l), 1.5 µl MgCl₂, 1 µl cDNA and sterile distilled water (final volume of 25 µl). RASSF1A-specific primers were used to achieve PCR amplification. GAPDH was selected as a reference owing to its stable expression (8). RASSF1A primers were selected from a previously published study (9). The primers used were: forward: 5′-GGCGTCGTGCGCAAAGGCC-3′ and reverse: 5′-GGGTGGCTTCTTGCTGGAGGG-3′. The primer sequences for GAPDH were: forward 5′-ACCACAGTCCATGCCATCAC-3′ and reverse 5′-TCCACCACCCTGTTGCTGTA-3′. The PCR amplification step consisted of initial denaturation at 95°C for 5 min, 35 cycles of denaturation at 94°C for 30 sec, annealing at 56°C for 30 sec, extension at 72°C for 60 sec and a final extension at 72°C for 5 min. Thus the PCR amplicons were visualized on 2% agarose gel (Novelab, Shanghai, China).