Mouse actb mm02619580 g1

qPCR was performed in 10 μl final reaction volumes containing the one-step master mix gb Ideal PCR Master Mix (Generi Biotech, Czech Republic), qPCR was performed by a LightCycler 96 (Roche, Switzerland) with preheating at 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 62°C for 1 min. Osteocalcin expression levels (Mouse Bglap, Mm03413826_mH, TaqMan Gene Expression Assay, Thermo Fischer, USA) were calculated using the ΔΔCT method, with normalisation against actin levels (Mouse Actb, Mm02619580_g1, TaqMan Gene Expression Assay, Thermo Fischer, USA), which was used as the internal control. For both groups, analysis was performed in three biological replicates, reactions were performed in triplicates for each sample. Statistical analysis was performed for both groups (t-test, p ≤ 0.05).

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^{101 (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).