Pcr primer

To analyse the relative expression of different mRNAs, the amount of cDNA was normalized based on signals from the ubiquitously expressed GAPDH [9 (link), 24 (link)]. Total RNA was extracted from cultured cells by using TRI Reagent (Molecular Research Center Inc., Cincinnati, OH, USA) and reverse-transcribed to cDNA using Reverse Transcription System (Takara Bio Inc., Shiga, Japan). PCR primer (Bioneer Co., Daejeon, Korea) pairs for human were selected to discriminate between cDNA and genomic DNA by using individual primers specific to different exons, when possible. Amplification was carried out at 94°C for 2 min, followed by 35 cycles at 94°C for 1 min, at appropriate annealing temperatures for each primer for 1 min, and at 72°C for 1 min [9 (link)]. Forward and reverse PCR oligonucleotide primers selected to amplify the cDNA are listed in Table 1 and RT-PCR products were separated electrophoretically on 2% agarose gels (Sigma Chemical Co.).

Plasma samples were purchased from Zen-Bio, Inc. (Research Triangle, NC, USA). Unless otherwise specified, a plasma volume of 1 mL was used for downstream analysis. DTBP, DMA, DMS, and DMP used for cfDNA extraction, as well as all other chemicals, were purchased from Sigma-Aldrich (St. Louis, MO, USA). Cell-free DNA ScreenTape Analysis and the 4200 TapeStation System used for cfDNA analysis were purchased from Agilent Technologies (Santa Clara, CA, USA). The DeNovix dsDNA Ultra High Sensitivity Assay Evaluation Kit and DS-11 FX+ spectrophotometer used for DNA analysis were purchased from DeNovix (Wilmington, NC, USA). The QIAamp Circulating Nucleic Acid Kit used for cfDNA extraction was purchased from QIAGEN (Dusseldorf, Germany). The AccuPower 2X GreenStar qPCR Master Mix, PCR Primer, lambda DNA, and the AccuPrep PCR/Gel Purification Kit were purchased from Bioneer (Daejeon, Korea). The CFX96 Touch Real-Time PCR Detection System used for real-time PCR was purchased from Bio-Rad Laboratories (Hercules, CA, USA). The PCR tubes and caps used for real-time PCR were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Experimental equipment, such as pipettes, was purchased from Eppendorf (Hamburg, Germany).

FD (NIES CRM No. 28) was obtained from the National Institute for Environmental Studies (Ibaraki, Japan). Dulbecco’s Modified Eagle Medium (DMEM), Ham’s F-12 nutrient mix, Fetal bovine serum (FBS), and penicillin-streptomycin mixture were purchased from GIBCO INC., NY, USA. Ethidium bromide, acridine orange, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 2′ 7′-dichlorodihydrofluorescein diacetate (DCF-DA), Triton™ X-100, Hoechst 33342, and paraformaldehyde were obtained from Sigma-Aldrich Co (St. Louis, MO, USA). SuperSignal™ West Pico PLUS Chemiluminescent Substrate was purchased from Thermo Fisher Scientific (Rockford, IL, USA). Ace-α-^® cDNA synthesis kit was purchased from ReverTra (Toyobo, Osaka, Japan). PCR primers were purchased from Bioneer Inc. (Daejeon, South Korea). Primary and secondary antibodies for western blot analysis, goat serum, Prolong^® Gold AntiFade Reagent with DAPI, and DyLight™ 554 Phalloidin were bought from Cell Signaling Technology (Beverly, MA, USA).