Sybr green qpcr mix

Tumor tissues were grinded in liquid nitrogen treated mortar, and total RNA was extracted by TRIzol according to the manufacturer’s instruction. The RNA was reversely transcribed to cDNA. RT-qPCR experiment was performed using SYBR Green qPCR Mix according to the manufacturer’s instruction on Roche LightCycler 96 instrument. Primers sequences used in this study were listed in table S2.

For RNA extraction and purification, cells or RNA-IPed material were mixed with TRI or TRI LS Reagent (Sigma) and purified on affinity columns using the Direct-Zol RNA miniprep kit (Zymo) that includes a DNase-treatment step. For cDNA synthesis, 1 μg of purified RNA for first treated with DNaseI (NEB) and then reverse-transcribed using the Transcriptor first strand cDNA synthesis kit (Roche) with oligo(dT) or random hexamers primers. For qPCR analysis, the cDNA were typically diluted in water (1:5) and used as a template for SYBR Green based qPCR using the SYBR Green qPCR Mix (Roche) and gene specific primers (See Supplementary Table S2) in a Step One Plus Real-Time PCR System (Applied Biosystems). Each reaction was performed in technical duplicates or triplicates. Relative expression levels were calculated with the ΔCt and the data was normalized to GAPDH or GUSB. Relative expression levels were calculated with the formula 2^−(ΔCt), where ΔCt is Ct(RL or FL)-Ct(GAPDH) or Ct(endogenous targets)-Ct(GUSB) and Ct is the equivalent cycle number at which the chosen threshold is crossed.

Total RNA was extracted from S2 cells (at least 1 × 10⁶ cells) by TRIzol (TakaRa, 9108) according to the manufacturer’s instructions, resuspended in 50 μL of nuclease-free water, and RNA concentration was detemined by Nanodrop UV-Vis spectrophotometer (Thermo scientific, ND-1000).
Total RNA (2–3 μg) from each sample was used for cDNA synthesis using reverse transcriptase (RTase) (Promega, USA) and oligo dT₁₈ primer (Intergrated DNA Technologies, USA) at 42°C for 60 min in a 25-μL reaction volume. Real-time PCR was performed using an Applied Biosystems with an SYBR Green qPCR mix (Roche, USA) in 20-μL reactions containing 10 μL SYBR premix, 4 μL H₂O, 1 μL each of forward and reverse primer (10 pmol/μL), 2 μL of cDNA template (1:20 diluted in deionized H₂O), using the following program: 50°C for 2 min, 95°C for 10 min, followed by 40 cycles of 95°C for 15 s, 60°C for 1 min and the dissociation curve analysis. Three biological replicates and three technical replicates were performed. Relative gene expression was determined by the 2^−ΔΔCt method⁶⁰ (link) using rp49 as a reference gene. Primers used for qRT-PCR experiments are listed in Table S2. The qRT-PCR experiment was in compliance with the criteria of the MIQE guidelines.⁶¹ (link)

Real-time PCR (ABI 7500) was performed to detect expression of the lncRNA HOTAIR, PRC2 complex (EZH2, SUZ12 and EED), LSD1 complex (LSD1, REST and CoREST), and methyltransferase (DNMT3A and DNMT3B) genes. The total reaction volume was 25 µl, and included 12.5 µl SYBR-Green qPCR Mix (Roche), 1 µl cDNA, 0.75 µl each of forward and reverse primers at 10 µmol/l (Table I), and DEPC water to 25 µl. Cycling parameters were as follows: 50°C for 2 min for 1 cycle; 95°C for 2 min for 1 cycle; 95°C for 15 sec, then 60°C for 30 sec for 40 cycles; melting curve analysis at 95°C for l5 sec, 60°C for l min, 95°C for l5 sec, 60°C for l5 sec for 1 cycle. GAPDH was used as an internal reference. Quantification of mRNA expression levels was performed by comparison of Ct values. Copy numbers were normalized to GAPDH and the ΔCt of target genes calculated as the average Ct value of target genes - average Ct value of reference genes. The mean was obtained and the relative expression values calculated using the 2^−ΔCt method and differences compared.