Rt2 sybr green qpcr mastermixes

The mRNA level was measured using real-time polymerase chain reaction. Briefly, Total RNA was extracted from cultured cells using TRIzol Reagent (Thermo Fisher Scientific), and cDNA synthesis was performed using the QuantiTect Reverse Transcription Kit (Qiagen). The primers used were as follows: ABL1 sense: 5′-CATCACGCCAGTCAACAGTCT-3′ and antisense: 5′-ACACCCTCCCTTCGTATCTCAG-3′. GADPH sense: 5′-TGACTTCAACAGCGACACCCA-3′, antisense: 5′-CACCCTGTTGCTGTAGCCAAA-3′. The real-time PCR was carried out by using RT2 SYBR® Green qPCR Mastermixes (Qiagen) according to the manufacturer's instructions. All PCRs were performed in triplicate. ΔΔCt method was used to calculate the relative expression levels.

Total mRNA was isolated using TRIzol Reagent (ThermoFisher, Waltham, MA, USA) and cDNA was synthesized using the RT² First Strand Kit (Qiagen, Hilden, Germany). qPCR reactions were prepared using RT² SYBR Green qPCR Mastermixes (Qiagen, Hilden, Germany) and performed using the CFX Connect Real-Time System (BioRad Laboratories, Hercules, CA, USA). Data was analyzed using the ΔΔCT method. Gene expression values were normalized against GAPDH expression. For fibroblast analysis, gene expression values were calculated as fold-change compared with expression level in fibroblasts derived from a 24-year old male control subject. For retinal organoid analysis, gene expression values were calculated as fold-change compared with the mean expression value derived from three cadaveric human retinal samples from working aged adults (28–60 years old). For RPE analysis gene expression values were calculated as fold-change compared with control iPSC-derived RPE. Primers used in this study are listed in Supplementary Table S1.

RHG epithelium was removed from the collagen hydrogel, lysed and total RNA was isolated using a RNA/Protein preparation kit (Qiagen, Hilden, Germany). Genomic DNA elimination and cDNA synthesis were performed using a reverse transcription kit (Qiagen) following the manufacturer’s guideline. Real-time PCR was performed using RT² SYBR® Green qPCR Mastermixes (Qiagen) with paired-primers for human beta defensin 1-3 (HBD 1-3; HP208395, HP208178, HP213186), adrenomedullin (ADM; HP205068), cathelicidin antimicrobial peptide (CAMP; HP207673) or housekeeping gene HPRT1 (HP200179), all purchased from OriGene Technologies, Rockville, USA. Briefly, 2 µl cDNA was added to 1 µl paired-primer, 9.5 µl nuclease-free water and 12.5 µl of SYBR green mastermix. The cycle threshold value was defined as the number of PCR cycles where the fluorescence signal exceeds the detection threshold value. Normalized by the expression of housekeeping gene HPRT1, the targeted mRNA induction was calculated by the ∆∆C_T analysis method following the formula: $$2^{-\mathrm{\Delta }\mathrm{\Delta }\mathrm{C}\mathrm{T}}=\frac{2^{-[\text{CT}(\mathrm{t}\mathrm{a}\mathrm{r}\mathrm{g}\mathrm{e}\mathrm{t})-\text{CT}(\mathrm{H}\mathrm{P}\mathrm{R}\mathrm{T}1)]}\mathrm{e}\mathrm{x}\mathrm{p}\mathrm{r}\mathrm{e}\mathrm{s}\mathrm{s}\mathrm{i}\mathrm{o}\mathrm{n}}{2^{-[\text{CT}(\mathrm{t}\mathrm{a}\mathrm{r}\mathrm{g}\mathrm{e}\mathrm{t})-\mathrm{C}\mathrm{T}(\mathrm{H}\mathrm{P}\mathrm{R}\mathrm{T}1)]}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}}$$

Keratinocytes cultured in flasks (passage 2) were harvested by trypsin treatment and were pelleted prior to RNA isolation. Keratinocytes cultured in multiwell dishes (passage 3) were lysed without trypsin treatment. Total RNA was purified using RNeasy Mini Kits (Qiagen Inc., Valencia, CA). RNA samples were treated with DNase I (Qiagen, Inc.) prior to synthesis of cDNA using the SuperScript^® VILO cDNA Synthesis Kit (Invitrogen/Thermo Fisher Scientific). Quantitative PCR (qPCR) was performed using gene-specific primers (RT² qPCR Primer Assays; Qiagen, Inc.), RT2 SYBR^® Green qPCR Mastermixes (Qiagen, Inc.) and the iCycler iQ system (BioRad, Hercules, CA). Technical triplicates were analyzed for each RNA sample, in addition to biological replicates. Expression levels were referenced to the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene using the comparative 2^−∆∆Ct method [38 (link)]. For comparison of mean expression levels in normal and keloid keratinocytes (eight cell strains each), gene expression levels were normalized to mean expression in normal keratinocytes. For comparisons of mean expression levels in treated and untreated cells (four cell strains per experiment), expression levels were normalized to mean expression in untreated cells.

A comparative CT method was used and for every plate a standard curve was set with 5 dilutions for target gene and reference gene. A comparison of all the time point samples with NF54 Time point 1 (2 hour) was made against a pooled reference comprised of NF54 RNA samples from 5 time points. Genes used in previous studies were used as endogenous control genes as reference, seryl-tRNA synthetase (PF07–0073) and actin (PFL2215w)37 (link), and the qRT-PCR was completed using Agilent Mx3000P qPCR System in reactions of 20 μl volumes using RT2 SYBR Green qPCR Mastermixes (Qiagen). The cycling conditions were 95 C for 15 min followed by 40 cycles of 94 C for 30 s, 54 C for 40 s and 68 C for 50 s with a final extension at 68 C for 10 min.