Rneasy mini kit isolation kit

Total RNA was isolated from PBMC lysates using the #RNeasy Mini Kit isolation kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. Quantity and quality of RNA assessments were performed using a UV/VIS spectrophotometer, the NanoDrop 2000c (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The RNA was reverse transcribed using High-Capacity RNA-to-cDNA kit (Applied Biosystems, Waltham, MA, USA; Thermo Fisher Scientific, Inc.) according to the manufacturer’s instructions. SNCA Hs00240906 gene expression and housekeeping gene (GAPDH Hs02786624, Thermo Fisher Scientific) were analyzed with Taq probe (TaqMan^TM Universal Master Mix, with UNG, Applied Biosystems; Thermo Fisher Scientific, Inc.) using StepOne Plus Real-Time System (Applied Biosystems; Thermo Fisher Scientific, Inc.). Each sample was analyzed in triplicate.

Isolation of total RNA from mouse brain and cerebrocortical cell cultures as well as qRT-PCR were performed and analyzed using the ∆∆Ct approach as previously described15 (link)79 (link). Briefly, following treatment, cerebrocortical cells were transferred onto ice, quickly washed once with cold 1 x PBS and total RNA was purified using an RNeasy Mini Kit isolation kit (Qiagen, Valencia, CA, cat# 74104), according to the manufacturer’s instructions. Total mouse brain RNA was isolated using the Qiagen RNeasy Lipid Tissue Midi Kit (Qiagen, cat# 75144). A list of the qRT-PCR primers is provided in Table 1.

Lymphocyte isolation was performed from 5 mL of venous blood, diluted 1:1 with phosphate buffered saline (PBS) (Sigma, St. Louis, MO, USA)/2% fetal bovine serum (FBS) (ThermoFisher Scientific, Waltham, MA, USA). Subsequently, 4 mL of Ficoll-Paque (Gibco ThermoFisher Scientific, Waltham, MA, USA) were added. It was centrifuged at 200× g, and the opaque interface of interest was removed. From 2 × 10⁶ lymphocytes, RNA extraction was performed using an RNeasy Mini kit isolation kit (Qiagen, Hilden, Düsseldorf, Germany), following the manufacturer’s recommendations. RNA extraction was performed just after lymphocyte isolation, aliquoted, and stored at −70 °C until further use. The RNA was quantified, and its integrity was determined from 5 µg in a 1% agarose gel with ethidium bromide and tris-acetate-EDTA buffer visualized in a Kodak Molecular Imaging Software (v.4.5.1) imaging system, while its purity was calculated using the A260/A280 ratio. All the RNA samples used in the present study were considered intact and with an optimal purity value.