Validated RT-qPCR Primer Sequences for Gene Expression Analysis

Forward and reverse primer pairs for SYBR-Green-based RT-qPCR analysis were designed in-house and validated under stringent conditions (efficiency between 90–110% and R² > 0.99), as described previously [55 (link),69 (link),70 (link),71 (link)]. Table 1 lists the validated RT-qPCR primer sequences utilized in this study. RT-qPCR reactions were performed using the QuantStudio 5 Real-Time PCR instrument (ThermoScientific) in 96-well plates. The RT-qPCR reaction mix was completed in 15 μL reaction volume consisting of 5 ng cDNA, 600 nm forward and reverse primers, and 7.5 μL 2× Luna Universal qPCR Master Mix (NEB, M3003). The RT-qPCR cycling parameters consisted of 95 °C for one min followed by a two-step denaturation and extension cycle of 95 °C for 15 s and 60 °C for 30 s for a total of 40 cycles. Plate reading was performed at the end of the extension phase. A DNA melt curve was performed at the completion of each RT-qPCR experiment to assess the amplification specificity. The RT-qPCR data was analyzed using the 2^−∆∆Ct analysis method, as described previously [72 (link),73 (link)]. All samples were normalized to two independent housekeeping genes (RSP18 and RPL4). The relative mRNA expression of each gene was reported as the mRNA fold change ± standard error of means (SEM) relative to the CGL1^dCas9 control cells.