7900ht fast real time pcr

Expression levels of candidate differentially methylated genes identified from our array, GTF2H4, SKI, RIC3, EIF2AK3, GRIA4, and DMR gene TNXB were determined by qRT-PCR (delta delta Ct) using 7900HT Fast Real-Time PCR (Thermofisher Scientific, MA, USA). Total RNA was extracted from RPE isolated from four AMD-affected donors (excluding CNV) and three normal donors (Additional file 1: Table S2) from LEITR, Florida, using Direct–Zol RNA MiniPrep kit (Zymo Research, CA, USA). Two micrograms of total RNA was reverse transcribed into cDNA using Superscript III First-Strand cDNA Synthesis (cat no. 18080-051, Thermo Fisher Scientific, MA, USA) and equal amounts of cDNA (30 ng) amplified using Power Up SYBR Green with gene-specific primers (Additional file 1: Table S6). GAPDH was included as an endogenous control. Fold change was calculated between different subgroups using the delta delta Ct method [28 ] and significantly differentially expressed genes were identified based on a p value< 0.05. Experiments were performed in triplicate and data pooled to generate normal versus AMD expression changes.

Complementary deoxyribonucleic acid (cDNA) was obtained by a reverse transcription of the RNA extracted using a high-capacity complementary DNA reverse-transcription kit (Thermo Fisher, Madrid, Spain). Quantitative polymerase chain reactions (qPCRs) were performed in 384-well plates in a 7900HT fast real-time PCR (Thermo Fisher, Madrid, Spain) using iTaq™ Universal SYBR^® Green Supermix (Bio-Rad, Barcelona, Spain). The thermal-cycle program used in all qPCRs was 30 s at 90 °C, 40 cycles of 15 s at 95 °C and 1 min at 60 °C. The analyzed liver genes were normalized by the housekeeping gene peptidylprolyl isomerase A (Ppia). The primers used for each gene were obtained from Biomers (Ulm, Germany) and can be found in Table 1. The relative expression of each gene was calculated using the 2^-∆∆Ct method, as reported by Schmittgen and Livak [38 (link)].

TRIzol (Ambion, United States) was utilized to extract total RNA. The SYBR Green qPCR Master Mix (MedChem Express, NJ, United States) was utilized for all RT‐qPCR analysis. The 7900HT Fast Real‐Time PCR (Thermo Fisher Scientific, MA, United States) was performed to evaluate amplification reactions. As a control, GAPDH and U6 were utilized for normalization. Relative gene expression was quantified using 2^−ΔΔCT method. All premiers were shown as shown in Table 2.