Power sybr green

Total RNA was isolated from mature cells (4–8 days) using a total RNA isolation kit (RNA-spin, iNtRON Biotechnology, Seongnam, Korea). RNA (1 μg) was converted to cDNA using Maxime RT premix (iNtRON Biotechnology). Power SYBR Green (Roche Diagnostics Gmbh, Mannheim, Germany) was employed to quantitatively determine transcription levels of genes by quantitative RT-PCR (Stratagene 246 mix 3000p QPCR System, Agilent Technologies, Santa Clara, CA, USA). PCR reactions were run in duplicates for each sample. Transcription levels of all genes were normalized to the level of β-actin. Sequences of primer sets used in this study are listed in Table 1.

Total RNA was extracted using the Direct‐zol RNA MiniPrep Kit (Zymo Research). The RNA was reverse transcribed using iScript Reverse Transcription Supermix for RT‐qPCR according to the manufacturer's instructions (Vazyme). The primer sequences are shown in Table S4. RT‐qPCR was performed in a StepOnePlus Real‐Time PCR System using Power SYBR Green (Roche). Relative RNA‐expression levels were calculated using the comparative Ct method and normalized to actin mRNA expression. Data are expressed relative to a calibrator using the 2^−ΔΔCt method.

Total RNA was isolated from patient samples using the RNeasy mini Kit (Qiagen). RNA was treated with RNase-free DNase set (Qiagen) to remove contaminating genomic DNA. The cDNA was synthesized using the Transcriptor High Fidelity cDNA Synthesis Kit (Roche Applied Science), quantified by Power SYBR Green (Roche Applied Science) or TaqMan qPCR. For SYBR green analyses, signals were detected with a CFX96 Real-Time PCR Detection System (Bio-Rad). Primers used for gene amplification were designed using Primer3 software and are listed in the table below. Relative expression levels were determined by the delta Ct method and expression level of HPRT was used for normalization. For TaqMan method, PCR Master Mix (Thermo Fisher) was used and signal detected with 7500 Fast Real-Time PCR System (Applied Biosystem). Probes used for TaqMan analyses were HIST1H1D: Hs00271187_s1; HIST1H2BG: Hs00374317_s1; HIST1H2BH: Hs00374322_s1; PGK1: Hs00943178_g1; PPIA: Hs04194521_s1 (Thermo Fisher). Relative expression levels were determined by the delta Ct method, taking the mean expression level of PKG1 and PPIA for normalization [44 (link)].