RNA extraction and RT-qPCR analysis

Total RNA was extracted using TRIzol reagent (Thermo Fisher Scientific, Inc.). For RNase R digestion, total RNA was incubated with 3 U/mg RNase R (Epicenter, Madison, WI, U.S.A.) for 15 min at 37°C. cDNA was synthesized using PrimeScript RT reagent Kit (TaKaRa, Dalian, China). The synthesized cRNA were then used for qPCR analysis with the Power SYBR Green Master Mix (Applied Biosystems, Foster City, CA, U.S.A.) using 1 μl cDNA as template on a 7500 Real-time PCR System (Applied Biosystems). Relative gene expression was calculated using 2^−ΔΔC_t method [6], with the housekeeping gene GAPDH or U6 as an internal control.