Realplex mastercycler 2

Total RNA from the CEPO-treated and nontreated HUVECs were extracted using Trizol^® RNA isolation reagent (Invitrogen, Waltham, MA, USA) according to the manufacturer’s instructions. NanoDrop ND-1000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA) was used to determine the concentration and purity of RNA at 260/280 nm. For the quantitative gene expression analyses, 500 ng RNA was reverse transcribed into cDNA (Applied Biosystems High-Capacity cDNA Reverse Transcription Kit, Foster City, CA, USA) using thermal cyclers (Techne Cole-Parmer, Vernon Hills, IL, USA). Gene expression analyses were performed by quantitative real-time PCR (applied biosystems QuantStudio 5 and Eppendorf Mastercycler Realplex 2) using 500 nM of each forward and reverse primers (Supplementary Table S1) and SYBR green Universal PCR master mix (Gendepot, Baker, TX, USA and Applied Biosystems, Waltham, MA, USA). Amplification conditions are 40× cycles of 94.0 °C for 2 s, 60.0 °C for 30 s, and 72.0 °C for 30 s. Primers for the amplification of gene targets were designed using the Primer3 program (https://bioinfo.ut.ee/primer3-0.4.0/, accessed on 20 October 2022). The expression level of each gene target was normalized with the housekeeping gene (Glyceraldehyde 3-phosphate dehydrogenase (GAPDH)), and the fold change of transcription was quantified using the relative quantification 2^−∆∆Ct method.

Ten genes were chosen from microarray analysis to be validated by qPCR namely, SYPL1, RAB5, AK5, PICALM, CAP8AP2, APOE, GABARAPL1, PSEN1, CREB1 and ADAMTS5. The reverse transcription kit was used to synthesize the first-strand cDNA (ReverTra Ace qPCR RT Master Mix with gDNA Remover (Code No. FSQ-301). Real-time PCR was carried out using PrimeTime^® Gene Expression Master Mix (IDT, USA) at 1X concentration containing PrimeTime^® qPCR primers and 3 pg to 100 ng cDNA template. PrimeTime Standard qPCR Assay (5′–3′Dye-Quencher Mod: 6-FAM/ZEN/IBFQ) primers (GAPDH and ACTB) were used as an endogenous control to quantify the target genes. The final volume of each RT-qPCR reaction was 20  µL, which contained 10  µL PrimeTime^® Gene Expression Master Mix (IDT, USA), 1 µL of each PrimeTime^® qPCR Assay primer (IDT, USA), 2 µL of diluted cDNA template and 7  µL of nuclease free water. PCR cycling protocol was performed by using Eppendorf Mastercycler Realplex2 (Eppendorf, Germany). Cycling included polymerase activation step of 3 min at 95 °C was followed by 45 cycles of 5 s at 95 °C and 30 s at 60 °C. Data analysis on expression levels were calculated using the 2^−ΔΔCt comparative CT method (Schmittgen & Livak, 2008 (link)). The means and standard deviations were calculated from experiments performed in triplicate and are presented as the n-fold differences in expression.