Mulv reverse transcriptase

To evaluate the expression of different AQP genes, OA chondrocytes (n=4) were cultured in 6-well plates with DMEM for 12 h, and RNA was extracted using the QIA shredder and RNeasy Mini kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. For RT-PCR, 1 µg of total RNA was reverse transcribed to first-strand complementary DNA (cDNA) using 1.25 µM oligo-dT primers in 40 µl PCR buffer II containing 2.5 mM MgC1₂, 0.5 mM dNTP mix, 0.5 U RNase inhibitor, and 1.25 U MuLV reverse transcriptase (PerkinElmer, Inc., Foster City, CA, USA) at 42°C for 60 min. The cDNA amplification was performed under the following PCR conditions: 94°C for 5 min; followed by 35 cycles of 94°C for 30 sec, 55°C for 30 sec, and 72°C for 30 sec; and a final extension at 72°C for 2 min using AmpliTaq Gold DNA Polymerase (cat. no. N8080241; Thermo Fisher Scientific, Inc., Rockford, IL, USA) and specific primers (Table I). The amplicons, along with the TrackIt 50 bp DNA ladder (cat. no. 10488-043; Thermo Fisher Scientific, Inc.), were resolved using 3% polyacrylamide gel electrophoresis, and the signals were visualized using the LAS-3000 mini system (FujiFilm, Tokyo, Japan).