Apr 02, 2023
qPCR of α-synuclein, TNF and NF-κβ
- 1Department of Clinical and Movement Neurosciences, UCL Queen Square Institute of Neurology, London, UK;
- 2Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, Maryland, USA
Protocol Citation: Michael J Hurley 2023. qPCR of α-synuclein, TNF and NF-κβ . protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzb2z2vx1/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 27, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 77702
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson’s
Grant ID: ASAP-000420
Abstract
A procedure for quantitative real time reverse transcription PCR of α-synuclein, TNF and NF-κβ
Materials
β-actin
Forward 5’-GCTGTCCCTGTATGCCTCTG-3’
Reverse 5’-GATGTCACGCACGATTTCCC-3’
α-synuclein
Forward 5’-CAGAGGCAGCTGGAAAGACA-3’
Reverse 5’-CACCACTGCTCCTCCAACAT-3’
TNF
Forward 5’-GAGCCAGCGCGCCAACGCCCTCCT-3’
Reverse 5’-TGAGGAGCACGTAGTCGGGGCAGC-3’
NF-κβ
Forward 5’-ACTGCCGAGCTCAAGATCTGCCGA-3’
Reverse 5’-AAGGAGCCTCGTGCCTCCCAGCCT-3’
qPCR
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.