Tri rna reagent

The RNA isolation of α-syn-transfected RA-differentiated cholinergic cells was performed by first dissolving the cells in 200 µL of Tri-RNA reagent (Sigma–Aldrich, St Louis, MO, USA; Cat. #: T9424). Chloroform was then added in a 1:5 ratio and shaken to mix thoroughly, followed by centrifugation at 12,000 g, 4 °C for 15 min. The aqueous phases were carefully removed into fresh centrifuge tubes, and isopropyl alcohol was then added at a 1:2 ratio. The mixtures were next incubated at RT for 10 min, followed by centrifugation at 12,000×g, 4 °C for 15 min. The supernatants produced were consequently discarded while the pellets were rinsed with 75% ethanol, followed by centrifugation at 7500 g, 4 °C for 5 min. The pellets were then air-dried for 5 min and subsequently dissolved in 30 µL of Milli-Q water. The purity of the RNAs extracted was determined using the nanodrop-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). RNA isolations (initial input amounts of 1000 ng) with a 260/280 value of above 1.90 were deemed as pure and reverse transcribed into cDNAs using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA; Cat. #: 4374966).

Total RNAs from tissues were extracted using Tri-RNA reagent from Sigma (St Luis, MO) according to the manufacturer’s instruction and 1 microG of total RNA was reverse transcribed into cDNAs using the first-strand synthesis kit (Roche, Tucson, AZ, USA). Real-time quantitative PCR analyses were performed according to a previously published procedure [47 (link)]. Triplicate CT values were analyzed in Microsoft Excel using the comparative Ct(DDCt) method as described by the manufacturer (Applied Biosystems, Foster City, CA, USA). The amount of target (ddCt) was obtained by normalization to an endogenous reference (Gapdh for mice) and relative to a calibrator. All TaqMan primers and probes were purchased from Applied Biosystems Inc.

Total RNAs of cells were extracted using Tri-RNA reagent from Sigma according to the manufacturer’s instruction and 1 μg of total RNA was reverse transcribed into cDNAs using the first-strand synthesis kit (Roche, Tucson, AZ, USA). Real-time quantitative PCR analyses were performed according to a previously published procedure. Triplicate C_T values were analyzed in Microsoft Excel using the comparative C_t(ΔΔC_t) method as described by the manufacturer (Applied Biosystems, Foster City, CA, USA). The amount of target (2^−ΔΔCt) was obtained by normalization to an endogenous reference (Gapdh for mice and GAPDH for humans) and relative to a calibrator. All taqman primers and probes were purchased from Applied Biosystems Inc. and the catalog numbers are shown below (Table 1):