qPCR of α-synuclein, TNF and NF-κβ

Extract total RNA and protein from cells or tissue using TRI Reagent® solution according to the manufacturer's instructions. Use RNase free glycogen as a carrier for the total RNA.

Keep protein for use in western blot.

Dissolve the RNA in nuclease free water.

Measure the concentration of RNA with a spectrophotometer (e.g. Nanodrop 1000).

Make cDNA from 2 micrograms of total RNA with a high-capacity cDNA reverse transcription kit (Invitrogen) according to manufacturer's instructions.

Dilute cDNA from the reverse transcription reaction 1:10 with nuclease free water.

Amplify 2 microliters of cDNA with gene specific intron-spanning primers (see materials) using PowerUp™ SYBR™ Green Master Mix (Invitrogen) according to the manufacturer's instructions with a StepOne Real-Time PCR system (Applied Biosciences).

Analyse using the using the comparative 2^-ΔΔC_T method.