Total rna mini plus concentrator kit

Total RNA was extracted from cells using Total RNA Mini Plus Concentrator kit (A&A Biotechnology, Gdansk, Poland). For assessing RNA quality and yield, A260/A280 and A260/A230 ratios for RNA preparation samples were analyzed with a DeNovix DS-11 FX+ spectrophotometer (DeNovix Inc., Wilmington, DE, USA). To determine Parkin mRNA expression, reverse transcription was carried out using High-Capacity RNA-to-cDNA Kit (Applied Biosystems) with total RNA (2 μg) according to the manufacturer’s instructions. Real-time PCR was performed in triplicates using TaqMan Gene Expression Assay probes for Parkin (Rn00571787_m1), Gapdh (Rn01775763_g1) and β-actin (Rn01412977_g1) with TaqMan Fast Advanced Master Mix (Applied Biosystems) in the ABI 7500 FAST Real-time PCR System (Applied Biosystems). The thermal cycling was initiated by the polymerase activation step for 2 min at 95 °C, followed by 40 cycles at 95 °C for 3 s and 60 °C for 30 s. mRNA levels of β-actin or Gapdh were used as an internal control to normalize the mRNA levels of Parkin. The expression levels for Parkin were assessed in relation to Gapdh and β-actin expression (ΔΔCt method, according to Livak and Schmittgen (2001) [42 (link)]).

The cells were proceed as described in Whole cell fluorescence except after 1 h incubation with appropriate inducer cellular RNA was extracted with Total RNA Mini Plus Concentrator Kit (A&A Biotechnology) according to the manufacturer’s instructions. For the mRNA stability experiment culture samples were taken starting 30 s prior to addition of rifampicin (250 μg/ml, BioShop, Burlington, Canada). Culture sample volumes were corrected for OD to maintain similar cell numbers per sample. Samples were immediately mixed with 0.5 ml of stayRNA protection buffer (A&A Biotechnology). cDNA were obtained after RNase-free DNase I (Thermo Scientific) treatment by using RevertAid First Strand cDNA Synthesis Kit (Thermo Scietific).

The fresh hippocampal sections were homogenized in Fenozol reagent and stored at −80 °C. Total RNA was purified using the Total RNA Mini Plus Concentrator Kit (A&A Biotechnology, Gdansk, Poland), according to the manufacturer’s instructions. To evaluate RNA quality and yield, the A260/A280 and A260/A230 ratios for RNA samples were analyzed with a DeNovix DS-11 FX+ spectrophotometer (DeNovix Inc., Wilmington, DE, USA). cDNA synthesis was performed using a high-capacity RNA-to-cDNA kit (ThermoFisher Scientific, Waltham, MA, USA), following the manufacturer’s instructions. 2 µg of RNA were used per 20 µL total reaction volume. Transcription was performed at 37 °C for 60 min and inactivated at 95 °C for 5 min. The cDNA was stored at −20 °C until it was used.