Actb vic mgb

RNA was isolated using Qiagen RNEasy Mini Kit, with RNA input normalized across samples before cDNA reaction. Samples were pretreated with dsDNase and reverse transcribed using Maxima First Strand cDNA synthesis (Thermo Fisher Scientific). All qPCRs were analyzed using a Bio-Rad CFX96 96-well optical cycler. To assess SAMD9L expression, primers targeted either human SAMD9L mRNA (forward: 5′-CTG​GGA​TTC​TGA​GCA​TCC​TTT​C-3′, reverse: 5′-CAC​ATG​CTC​TTT​GGT​CCA​GT-3′) or SAMD9L plasmid (forward: 5′-GCT​GGG​CAG​AGT​CTC​CTA​AA-3′, reverse: 5′-CAC​AAG​GAG​AAG​CAC​AGG​AA-3′), and expression was compared with HPRT mRNA (forward: 5′-GTT​GGA​TAT​AAG​CCA​GAC​TTT​GTT​G-3′, reverse: 5′-AGG​GAA​CTG​ATA​GTC​TAT​AGG​CT-3′). qPCR was performed using Power SYBR Green (Thermo Fisher Scientific) and 400 nM primers and cycled according to manufacturer instructions. To assess mCherry and GFP mRNA expression, Taqman gene expression assays (Thermo Fisher Scientific) with Taqman Fast Advanced Master Mix (Thermo Fisher Scientific) were used according to manufacturer instructions. GFP FAM-MGB (assay ID: Mr04097229_mr; Thermo Fisher Scientific) or mCherry FAM-MGB (assay ID: Mr07319438_mr; Thermo Fisher Scientific) were multiplexed with ACTB VIC-MGB (assay ID: Hs01060665_g1; Thermo Fisher Scientific) as a loading control.