RNA purification and semi-quantitative RT-PCR

Total RNAs was extracted by TRIzol reagent (Invitrogen) according to the manufacturer's protocol, and subsequently treated with DNase I (Promega) at 37 ºC for 30 minutes, followed by 95 ºC for in-activation of DNase I. To digest linear RNA, 1 μg of total RNAs from 293T cells was subjected to 4 U RNase R (Geneseed) treatment in 20 μl reactions at 37 ºC for 20 minutes, followed by RNase R heat inactivation at 70 ºC for 10 min. 1 μg of RNA was applied to synthesize cDNA using Reverse Transcriptase (Takara) according to the manufacturer's instructions. The RT products were then diluted 1:20 used for PCR amplification. Semi-quantitative PCR was performed by incubation at 94 °C for 5 min followed by 25 cycles of 94 °C for 30 s, 61 °C for 30 s, and 72 °C for 10 s on a T100 PCR Thermal Cycler (Bio-Rad). RT-PCR products were then separated on 2.5% agarose gels running with 1× TAE buffer, and scanned with a scanner (Tanon, 2500R). The resulting bands intensity was quantified by Image J software. Real-time PCR was performed using Premix Pro Taq HS qPCR kit (AG11701). ΔΔCt (Cycle threshold) was calculated using housekeeping gene GAPDH. Statistical analysis was performed using a Student's t-test. The primers used in the semi-quantitative PCR and qPCR were listed in Table S1.