Genomic dna remover

Total RNA was extracted from siRNA-treated HSC-1 cells by RNAiso plus (Takara Bio Inc.). cDNA was prepared by ReveTra Ace qPCR RT Master Mix with genomic DNA Remover (Toyobo, Ltd.). qRT-PCR was performed with THUNDERBIRD SYBR quantitative PCR Mix (Toyobo, Ltd.) using StepOnePlus RT-PCR System (Applied Biosystems) according to the manufacturer’s protocol. Data were normalized with β-actin. Primers used for RT-PCR were collagen VII, 5′-GCTGGTGCTGCCTTTCTCT-3′ and 5′-TCCAGGCCGAACTCTGTC-3′; and β-actin, 5′-CCAACCGCGAGAAGATGA-3′ and 5′-CCAGAGGCGTACAGGGATAG-3′.

The total RNA was lysed in the TRIzol Reagent (Thermo Fisher, #15596026), followed by conventional chloroform extraction. 500 ng of total RNA was reverse transcribed using ReverTra Ace qPCR RT Master Mix with genomic DNA Remover (Toyobo, #FSQ-301). Realtime qPCR was performed using PerfectStart Green qPCR SuperMix (Transgen, #AQ601) in qTOWER 2.0 (Analytik Jena AG, Germany) with thermocycler setup: 95°C for 1 min, 40 cycles of 95°C for 15 s and 60°C for 45 s (plate read), followed by 60–95°C melting curve. Primers of ACTB, MT-CYB, or U6 that were used as internal control and all the lncRNAs and NATs were marked in Supplementary Figure S1C (Supplementary Table S3). All tests were measured triplicated. The 2^−ΔΔCt method was used to analyze and compare the fold change. Primer walking PCR was performed using PCR MasterMix (TIANYA BIO, #M0121-1ml): 95°C for 5 min; 35 cycles of 95°C for the 30 s and 61°C for 30 s, and 72°C for 2.5 min; and extra extension at 72°C for 10 min (Supplementary Table S3).

Human fibroblast BJ from neonatal foreskin and SKBR3 (breast adenocarcinoma, human) cell lines were purchased from ATCC. Fibroblast cells were grown in DMEM supplemented with 10% HyClone FBS, non essential amino acids, 100 U/ml penicillin, 100 μg/ml streptomycin, at 37°C in 5% CO₂. SKBR3 cells were grown in ATCC-formulated McCoy's 5a modified medium complemented with 10% FBS and were maintained under an atmosphere of 5% CO₂ at 37°C.
The effect of alkylating PIP 2 on the expression of ERBB2 mRNA was determined in both fibroblast and SKBR3 cell lines using real-time PCR. BJ skin fibroblast cells were seeded at a density of 5 × 10⁴ cells/well of a 6-well plate and SKBR3 cells were plated into the 6-well plate at 4 × 10⁵ cells/well. The cells were then treated with 50 and 100 nM of alkylating PIP 2 for 48 h with DMSO as a corresponding control sample. After 48 h, total RNA was isolated using RNEasy Kit (Qiagen) and cDNA was synthesized by ReverTra Ace qPCR RT Master mix with genomic DNA remover (Toyobo, Japan) following the manufacturer's instructions. The expression level of ERBB2 was normalized using β-actin, as an internal control.
The primers used in this study includes, β-actin sense, 5′-CAATGTGGCCGAGGACTTTG-3′ and antisense, 5′-CATTC TCCTTAGAGAGAAGTGG-3′. The sense primer of ERBB2 is 5′-AGCCGCGAGCA CCCAAGT-3′ and antisense, 5′-TTGGTGGGCAGGTAGGTGAGTT-3′.