Mesa blue qpcr mastermix plus kit for sybr assay low rox

To remove any remaining genomic DNA after RNA extraction, the independent set of 28 human donor samples were treated with DNA‐free DNA Removal Kit (Invitrogen, UK). Complementary DNA was then synthesised with First Strand cDNA Synthesis Kit (Life Technologies/Thermo Fisher Scientific, UK), following the manufacturer's standard protocols. qPCR was performed with technical triplicates using a modified protocol described previously²⁸ with MESA BLUE qPCR Mastermix Plus Kit for SYBR assay (Low ROX; Eurogentec, Southampton, UK) and a Stratagene MX3005P^® qPCR System (Stratagene, La Jolla, CA, USA). The primer sets (Table S1) were custom synthesized (Eurogentec, Southampton, UK). The melt curve analysis and agarose gel electrophoresis were used to confirm the specificity of amplification reactions. The comparative expression value of the gene of interest was calculated using the efficiency‐corrected ddCt method, with normalization against three house‐keeping genes (GAPDH, RPL5 and ACTB) for the in situ data and four house‐keeping genes (GAPDH, RPL5, ACTB and TUBB) for the hfRPE in vitro studies. All house‐keeping genes were selected and combined into a single standard using BestKeeper version1.²⁹

RNA isolation was carried out using the RNeasy Plus Mini‐Kit (Qiagen, Hilden, Germany). Complementary DNA was synthesized from RNA using the First Strand cDNA Synthesis Kit (Thermo Scientific, Waltham, USA). Quantitative PCR was performed with the MESA BLUE qPCR Mastermix Plus Kit for SYBR assay (Low ROX; Eurogentec, Belgium) using a modified version of a previous protocol.41 Reactions were run on a Stratagene MX3000P qPCR System (Stratagene, California, USA), with a minimum of three biological replicates for each experimental condition and three technical replicates for each cDNA sample. Primer sets used are listed in Table 2. Final values were expressed relative to a calibrator sample assigned an arbitrary value of 1 and normalized to the expression of three housekeeping genes, beta tubulin, GAPDH and ribosomal protein L5 using the efficiency‐corrected ddCt method. The specificity of amplification reactions was confirmed by melt curve analysis.