Stepone plus pcr instrument

Total RNA was extracted and purified using TRIzol (Invitrogen) according to the manufacturer’s instructions. The RNA concentration was identified by NanoDrop^TM 2000 UV-Vis Spectrophotometer (ThermoFisher, Waltham, MA, USA) following the manufacturer’s instructions. Complementary DNAs (cDNAs) were synthesized from l μg of total RNA by quantitative reverse transcription PCR (qRT-PCR) using the TaKaRa RNA PCR Kit Ver. 2.1 (TaKaRa Bio). Real-time qRT-PCR was performed with the SYBR Green real-time qPCR Kit (TaKaRa Bio) using a StepOnePlus PCR instrument (Applied Biosystem, Waltham, MA, USA). The PCR products were subjected to a melting curve analysis and the mRNA expression levels were calculated using the 2^−ΔΔCt method [20 (link)]. GAPDH expression levels were used as internal controls and to normalize the expression levels. The primers used for real-time qRT-PCR are shown in Supplementary Table S1 in page 1. All assays were repeated three times in parallel.

ChIP assays were performed as previously described25 (link) using the ChIP assay kit (EMD Millipore). Briefly, cells were cross-linked for 10 min at room temperature with 1% formaldehyde and their chromatin were sonicated (Bioruptor Plus, Diagenode) to obtain DNA fragments of 200–400 bp. Chromatin immunoprecipitations were performed with chromatin from 6 10⁶ cells (1 10⁶ from the epigenetic modifications) and 5 μg of antibodies (see Supplementary Table S2). Quantitative real-time PCR reactions were performed using 1/60^e of the immunoprecipitated DNA and the PerfeCTa SYBRgreen SuperMix (Quanta BioSciences). Relative quantification using standard curve method was performed for each primer pair and 96-well Optical Reaction plates were read in a StepOnePlus PCR instrument (Applied Biosystem). Fold enrichments were calculated as percentages of input values or as fold inductions relative to the values measured with IgG. Primer sequences used for quantification (see Supplementary Table S3) were designed using the software Primer 3.

Total RNA was isolated from cultured cells using an RNA isolation kit (Qiagen, West Sussex, U.K.) as recommended by the manufacturer. The isolated samples were treated with RNase-free DNase (Qiagen) to remove genomic DNA. Total RNA concentration and purity were measured at OD260 and OD260/280 ratio determined with NanoDrop ND-1000 spectrophotometer (NanoDrop Tech., Rockland, Del). One μg RNA was reverse-transcribed into double-stranded cDNA using High Capacity cDNA Reverse Transcription Kit (Thermo-Fisher/Applied Biosystems, Foster City, CA). TaqMan real-time PCR assay (Applied Biosystem) was performed in triplicates. Each reaction mixture contained 2μl cDNA mixed with 7 μl PCR grade water, 10 μl 2x TaqMan Universal PCR Master Mix, 1μl 20x PrimeTime qPCR assay kit (IDT, Coralville, IA) including forward and reverse primers and ZEN Double-Quenched FAM probes (Table 2). Parallel assays were done by detecting β-actin for normalization. PCR reactions were performed using StepOne Plus PCR instrument (Applied Biosystem) under the following parameters: 50°C for 2 min, 95°C for 5 min and 40 cycles at 95°C for 15 sec and 60°C for 1 min. After amplification, data of independent runs were analysed with the StepOne Plus Software v2.0.

Total RNA was extracted from the retinal tissue or cultured ARPE-19 cells using TRIzol reagent as per the manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA). cDNA was synthesized from 1.0 μg of total RNA using a Maxima first-strand cDNA synthesis kit (Thermo Fisher Scientific, Rockford, IL, USA) as per the manufacturer’s instructions. The quantitative PCR (qPCR) was performed to assess the expression of genes regulating inflammatory cytokines/chemokines, antimicrobial peptides, and intracellular junction proteins using the StepOnePlus PCR instrument (Applied Biosystems, Grand Island, NY, USA). Gene expression was analyzed by the threshold cycle (ΔΔC_T) method and normalized with Gapdh, an endogenous housekeeping reference gene.

To assess the ABCB1 gene copy number, TaqMan Copy Number Assay (Hs04939312_cn) was used. For the internal control RNaseP TaqMan Copy Number Reference Assay (assay ID 4403326) was selected. The assays were performed in duplicates using StepOne Plus PCR instrument (Applied Biosystems, Thermo Fisher Scientific). Reaction mix (20 μL) consisted of 1 × TaqMan Universal Master Mix II, no UNG, 2 × TaqMan Copy Number Assay, 2 × TaqMan Copy Number Reference Assay (all from Applied Biosystems, Thermo Fisher Scientific) and 20 ng of the DNA sample. Thermocycling parameters consisted of 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. Relative quantification analysis was done to estimate the ABCB1 copy number for each sample by using the Copy Caller Software v2.0 (Applied Biosystems).