After extraction of RNA from cells with TRI reagent (Invitrogen), TaqMan miRNA Reverse Transcription Kit (TMmRT Kit, Applied Biosystems, CA-USA) was employed to reversely transcribe the RNA into complementary DNA (cDNA).
Next, synthesis of the cDNA was accordingly performed using Prime-Script One Step cDNA Kit (Takara, Tokyo-Japan) and TMmRT Kit (Applied Biosystems, CA-USA). Through SYBR-Green Real Time PCR Kit (Takara) the reactions of qPCR were carried out on ABI Prism 7900HT (Applied Biosystems, CA-USA).
Composition of the reaction mixture (total volume of 20 µL) included cDNA (1 µL), forward primer (0.4 µL), reverse primer (0.4 µL), water of nuclease-free (8.2µL) and PerfectStart TM Green-Fluorescein qPCR SuperMix (10 µL, 2X).
Conditions of the reaction included 30 s at 95℃, then 10 s (40 cycles) at 95℃ and followed by 20 s at 58℃ and 30 s at 72℃. With mRNA of GAPDH as the internal control, the relative gene expression levels were computed using the 2-△△ Ct method.
Next, synthesis of the cDNA was accordingly performed using Prime-Script One Step cDNA Kit (Takara, Tokyo-Japan) and TMmRT Kit (Applied Biosystems, CA-USA). Through SYBR-Green Real Time PCR Kit (Takara) the reactions of qPCR were carried out on ABI Prism 7900HT (Applied Biosystems, CA-USA).
Composition of the reaction mixture (total volume of 20 µL) included cDNA (1 µL), forward primer (0.4 µL), reverse primer (0.4 µL), water of nuclease-free (8.2µL) and PerfectStart TM Green-Fluorescein qPCR SuperMix (10 µL, 2X).
Conditions of the reaction included 30 s at 95℃, then 10 s (40 cycles) at 95℃ and followed by 20 s at 58℃ and 30 s at 72℃. With mRNA of GAPDH as the internal control, the relative gene expression levels were computed using the 2-△△ Ct method.