Easy spin total rna purification kit

We performed RT-PCR and quantitative RT-PCR (qRT-PCR) as described previously^{31 (link)}; the primer sequences are listed in Supplementary Table 1. Briefly, we extracted total RNA using an Easy-Spin^® Total RNA Purification Kit (iNtRON Biotechnology, Seoul, Korea), and then converted 1 µg of RNA into cDNA with the iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA). QRT-PCR was performed using SYBR^® Premix ExTaq™ (TAKARA BIO Inc., Otsu, Japan) and the CFX96 Real-Time System (Bio-Rad). Beta-actin was used as an endogenous reference to calculate Ct values and relative expression levels (2^−ΔΔCt) of target genes. For semi-qRT-PCR, we performed PCR with the 2× EmeraldAmp® GT PCR Master Mix (TAKARA BIO Inc.) and 10 pmol of each primer (Supplementary Table 1). Amplified transcripts were normalized to the glyceraldehyde 3-phosphate dehydrogenase (GAPDH)-specific signal.

Total mRNA was extracted using an Easy-Spin Total RNA Purification Kit (iNtRON Biotechnology) and was used for first-strand cDNA synthesis using a PrimeScript RT Reagent Kit (Takara Bio) according to the manufacturer's instructions. Real-time qRT-PCR was performed using SYBR PremixExTaq (Takara Bio). Reactions were performed on a CFX96 Real-Time System (Bio-Rad), and the results were evaluated with CFX real-time detection system software. Quantification was performed by determining the threshold cycle value (C_T), and target genes were normalized to GAPDH (glyceraldehyde-3-phosphate dehydrogenase). Relative target gene expression was quantified using the comparative C_T method. All PCRs were performed in triplicate. Primer sequences are shown in Table S5.