Rnasin 1 plus rnase inhibitor

Total RNA was extracted using the Direct-zol RNA MiniPrep Kit (Zymo Research Corporation, Irvine, CA, USA) according the manufacturer’s instructions. Briefly, 1 μg of total RNA was incubated with 0.5 μg of oligo dT (MD.Bio., Taipei, Taiwan) at 70 °C for 15 min. Then, the RNA was mixed with buffer containing 0.25 mM dNTPs (MD. Bio., Taipei, Taiwan), 20 U of RNasin I Plus RNase Inhibitor (Promega, WI, USA) and 20 U of M-MLV Reverse Transcriptase (Promega) and incubated at 42 °C for 90 min for cDNA synthesis. This mixture was then used for specific cDNA amplification in a GeneAmp PCR system 2400 (Perkin Elmer, Waltham, MA, USA).
For mRNA quantification, Real-time PCR was performed using a standard LightCycler 480 SYBR Green I Master protocol on an LightCycler 96 System Roche, Basel, Switzerland). The 10 μl PCR included 2 μl RT product, 5 μl 2 × SyberGreen PCR Mix, 0.5 μl 10 μM forward primer, 0.5 μl 10 μM reverse primer and 2 μl ddH₂O. All reactions were run in triplicate. The cycle number at which the reaction crossed the threshold cycle (Ct) was determined for each gene and the relative amount of each gene to GAPDH was described using the equation 2^ΔCt where ΔCt=(Ct_{interested gene} – Ct_GAPDH).

Total RNA was extracted using the Direct-zol RNA MiniPrep Kit (Zymo Research Corporation, Irvine, CA, USA) according to the manufacturer's instructions. Briefly, 1 μg of total RNA was incubated with 0.5 μg of oligo dT (MD Bio., Taipei, Taiwan) at 70 °C for 15 min. Then, the RNA was mixed with buffer containing 0.25 mM dNTPs (MD Bio.), 20 U of RNasin I Plus RNase Inhibitor (Promega, San Luis Obispo, CA, USA) and 20 U of M-MLV Reverse Transcriptase (Promega) and incubated at 42 °C for 90 min to allow for cDNA synthesis. This mixture was then used for specific cDNA amplification in a GeneAmp PCR system 2400 (Perkin Elmer, Waltham, MA, USA).