M mlv rtase

Methods described in our previous report [35 (link)] were also used in performing total RNA preparation and cDNA synthesis. Monophasic solution of phenol and guanidine isothiocyanate (TRIzol Reagent, Invitrogen, Carlsbad, CA, USA) were used for the extraction of total RNA. RNA concentration was determined by a UV-visible spectrophotometer (Amersham Biosciences, Piscataway, NJ, USA) at 260 nm. Aliquots (1.0 µg) of RNA from each sample were reverse-transcribed using Moloney murine leukemia virus reverse transcriptase (MML-V RTase, Promega). 1× RT-buffer, 2 mM deoxynucleotide triphosphates (dNTPs, Promega), 0.2 pM oligo dT primer (16-mer) (Bioneer Inc., Daejeon, Korea), and MML-V RTase (2.5 units/µL) in 20 µL reaction volumes were added in performing reverse transcription. Then, the samples were incubated at 42 °C for 60 min and then stored at −20 °C.

Total RNA was prepared from cells using Trizol Reagent (Thermo Scientific, Seoul, Korea) according to the manufacturer’s instructions. Total RNA (1 μg/mL) and 50 μM oligo-dT primer were mixed in 15 μL of DEPC-water and reacted at 70 °C for 5 min. After the reaction, 2 μL of 100 mM dithiothreitol, 2 μL of 10 mM dNTP, 5 μL of 5× RT buffer, and 1 μL of 200 unit/μL M-MLV RTase (Bioneer, Daejeon, Korea) were added to synthesize cDNA in a reaction at 25 °C for 5 min, followed by 42 °C for 60 min and 70 °C for 15 min. Real-time quantitative PCR was performed on Rotor-Gene 6000 (Qiagen, Seoul, Korea) using SensiFast SYBR No-ROX kit, 10 pM of each primer (Supplementary Table S1), and 100 ng of cDNA. After amplification, the melting curve analysis was performed to confirm the specificity of the reaction. The end-point cycle threshold (Ct) used for real-time PCR quantification was defined as the number of PCR threshold cycles. The relative quantification of the target gene expression level was assessed using the ^ΔΔCt method.

Total RNA was isolated using TRIzol Reagent (Thermo Scientific, Seoul, Korea) ac-cording to the manufacturer’s instructions. cDNA was synthesized using M-MLV RTase (Bioneer, Daejeon, Korea) according to the manufacturer’s instructions. Real-time quantitative PCR was performed on a Rotor-Gene 6000 (Qiagen, Seoul, Korea) using the Sensi-Fast SYBR No-ROX kit and primers (Supplementary Table S1) according to the manufacturer’s instructions. Relative quantification of the target gene was calculated using the ^ΔΔCt method.