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Reverse transcription quantitative polymerase chain reaction (qRT-qPCR)

SL Shenglong Li
JL Jie Liu
SL Siyu Liu
WJ Weijie Jiao
XW Xiaohong Wang
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TRIzol reagent (Invitrogen, Carlsbad, CA) was used to extract the total RNA from chondrocytes or mouse knee cartilage. The Primescript RT kit (Takara, Chiga, Japan) was used for reverse transcription of cDNA according to the manufacturer’s instructions. Reverse transcription-PCR was performed using SYBR Green RT-PCR kit (Takara, Chiga, Japan). The samples were allowed to react at 37 °C for 15 min, 85 °C for 5 s and 4 °C for the remaining time. The cDNA generated following RT was maintained in a -80 °C refrigerator. The reaction conditions of qRT-PCR were set as follows: pre-denaturation at 95 °C for 30 s, with 40 cycles of denaturation at 95 °C for 10 s, annealing at 60 °C for 20 s, and extension at 70 °C for 10 s. GAPDH and U6 were used as the internal references. The primer sequences are listed in Table ​Table22.

Sequences of primers for qRT-PCR

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