Quantifying Ataxin-2 and TDP-43 Expression

At 48 hr after transfection, RNA was extracted from cells using the PureLink RNA Mini Kit (Invitrogen) and converted to complementary DNA (cDNA) using the iScript cDNA Synthesis Kit (Bio-Rad) according to the manufacturers’ instructions. qPCR was conducted using 20–40 ng of cDNA per reaction using iTaq Universal SYBR Green Supermix (Bio-Rad). Measurements for each biological replicate were conducted in technical triplicates using validated primers for ataxin-2^{97 (link)}. Values were compared to GAPDH, and the average fold-change was calculated using the 2^ΔΔCT method. All primer sequences are provided in Supplementary Table 1.
Tissues from injected animals were dissected and RNA was extracted and analyzed by qPCR as described above for ataxin-2. hTDP-43 mRNA was measured using the PrimeTime Gene Expression Master Mix (IDT) with the following TaqMan probes: human TDP-43 (Hs00606522_m1; ThermoFisher Scientific) and mouse Actb (Mm02619580_g1; ThermoFisher Scientific).