Tb green qpcr premix

Total RNA was isolated using RNAiso Plus (Total RNA extraction reagent, Takara, 9109) according to the manufacturers’ instructions and quantified by NanoDrop 2000 Spectrophotometer. One microgram of total RNA was reverse-transcribed to cDNA using PrimeScript RT Master Mix (Takara, RR036B). Twenty nanograms of DNA or cDNA templates were amplified using TB Green qPCR Premix (Takara, 639676) by QuantStudio^TM 12K Flex Real-time PCR system (Thermo Fisher Scientific). The relative expression levels of target genes were calculated with the △△ct method. All the primers sequences we used are listed in Supplementary Table 4.

The retina and RPE/choroid were dissected from at least three non-diseased human eyes of similar age (male and female) and cut into smaller pieces in cold PBS. The tissue samples were snap frozen in liquid nitrogen and stored at –80ºC for RNA preparation. Tissue samples (50–100 mg) were dissolved in 1 mL of Trizol reagent (Invitrogen, San Diego, CA). Total RNA was extracted using RNeasy mini kit as recommended (Qiagen, Valencia, CA). Complementary DNA was prepared using 1 μg of total RNA and the RNA to cDNA EcoDry Premix (TaKaRa, Mountain View, CA) and diluted 1:10. qPCR was conducted in triplicates using a Mastercycler Realplex (Eppendorf, Enfield, CT) and TB-Green qPCR Premix (TaKaRa). The cycles for amplification were 95ºC for 2 min; 40 cycles of amplification (95ºC for 15 s, 60ºC for 40 s); and dissociation curve step (95ºC for 15 s, 60ºC for 15 s, 95ºC for 15 s). The relative fluorescent units (RFUs) at a threshold fluorescence value (Ct) were used for linear regression line and assessment of nanograms of DNA. The target gene expression levels were determined by comparing the RFU at the Ct to the standard curve and normalized by the housekeeping gene ribosomal protein L13α (RPL13A). The primer sequences used in this study are listed in Table 1. Each sample was run in triplicates.

Total mRNA was isolated from A549 and THP1 cells using RNeasy^® plus Mini Kit (74104; Qiagen, Germany) as per manufacturer’s instructions. The cDNA was transcribed from 1 ug of mRNA using iScript cDNA synthesis kit (1708890; BIO-RAD, United States). The qPCR reactions were performed in triplicates using qPCR specific primers (Supplementary Table S1) using TB green qPCR premix (639676; TaKaRa, Japan) on a CFX96 Touch Real-Time PCR Detection System (BIO-RAD, United States). The fold change expression (-ΔΔCt) was calculated after normalization with β2-microglobulin (β2M) expression.