qPCR of α-synuclein, TNF and NF-κβ
Michael J Hurley
Abstract
A procedure for quantitative real time reverse transcription PCR of α-synuclein, TNF and NF- κβ
Steps
qPCR
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-ΔΔCT method.
