Diethylenepyrocarbonate treated water

Total RNA was isolated with TRIzol (Life Technologies, Carlsbad, CA, USA). The tissues in TRIzol were incubated for 10 minutes at 4°C. Furthermore, 100 μL of chloroform was added and gently mixed. After incubation for 10 minutes on ice, samples were centrifuged at 12,000 rpm for 15 minutes. Supernatants were mixed with 500 μL of isopropanol and incubated at 4°C for 15 minutes. After centrifuging at 12,000 rpm for 10 minutes, pellets were washed with 70% (v/v) ethanol. After allowing the ethanol to completely evaporate, pellets were dissolved in 100 μL of diethylenepyrocarbonate-treated water (Invitrogen Life Technologies, Carlsbad, CA, USA). Complementary DNA was prepared using reverse transcriptase originating from Murine Moloney leukemia virus (Promega, Madison, WI, USA), according to the manufacturer’s instructions. Polymerase chain reaction was performed for 35 cycles at 94°C for 30 seconds, 58°C for 30 seconds, and 72°C for 30 seconds. Oligonucleotide primers were Table 1.

After treatment, the medium was removed by suction, and cells were washed with Dulbecco’s PBS twice. RiboEX (500 μl; GeneAll, Seoul, Korea) was added to plates that were incubated for 10 min at 4°C. RiboEX was harvested and placed in a 1.5-ml tube, and 100 μl of chloroform was added and gently mixed. After incubation for 10 min in ice, samples were centrifuged at 10,000 × g for 30 min. Supernatants were extracted and mixed with 200 μl of isopropanol, and mixtures were incubated at 4°C for 1 h. After centrifuging at 13,000 × g for 30 min, the pellet was washed with 70% (v/v) ethanol. After allowing the ethanol to evaporate completely, the pellet was dissolved in 100 μl of diethylene pyrocarbonate–treated water (Invitrogen). cDNA was prepared using a reverse transcriptase originating from Murine-Moloney leukemia virus (Promega, Madison, WI), according to the manufacturer’s instructions. The polymerase chain reaction (PCR) primers used are shown in Supplemental Table 1*. PCR was performed using over 30 cycles of 94°C for 20 s, 58°C for 30 s, and 72°C for 45‍ ‍s. Oligonucleotide primers were purchased from Bioneer (Seoul, Korea).