Mar 12, 2025

Public workspaceiNeuron RNA-Extraction and RT-qPCR

DOI
dx.doi.org/10.17504/protocols.io.ewov1drryvr2/v1
  • 1Department of Neurodegenerative Diseases, UCL Queen Square Institute of Neurology, London, UK;
  • 2Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, USA, 20815
Benjamin O'Callaghan
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DOI: dx.doi.org/10.17504/protocols.io.ewov1drryvr2/v1
Protocol Citation: Benjamin O'Callaghan, Helene Plun-Favreau 2025. iNeuron RNA-Extraction and RT-qPCR. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov1drryvr2/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: March 04, 2025
Last Modified: March 12, 2025
Protocol Integer ID: 123793
Keywords: ASAPCRN, RT-qPCR, PINK1, iNeuron
Funders Acknowledgements:
Aligning Science Across Parkinson’s (ASAP)
Grant ID: ASAP 000478
Abstract
Protocol for the extraction of RNA from iNeurons and RT-qPCR measurements of PINK1 mRNA.
Materials
  • Monarch Total RNA Miniprep Kit (New England Biolabs, T2010S)
  • UltraPure DNase/RNase-Free Distilled Water (Invitrogen, 10977035)
  • SuperScript IV Reverse Transcriptase (Invitrogen, 18090050)
  • dNTP Mix (10 mM each) (Invitrogen, R0192)
  • Random Hexamers (50μM( (Invitrogen, N8080127)
  • RNaseOUT Recombinant Ribonuclease Inhibitor (Invitrogen, 10777019)
  • Power SYBR Green PCR Master Mix (Applied Biosystems, 4367659)
  • Gene Specific Primers (Sigma)
  • NanoDrop One Spectrophotometer (Thermo Fisher Scientific)
  • QuantStudio 7 Flex Real-Time PCR System (Applied Biosystems, RRID:SCR_020245)
RNA Extraction
RNA Extraction
iNeuron culture and initial collection for RNA extraction described in "iNeuron Differentiation and Culture in N2B27 vs BrainPhys for Immunofluorescence and Biochemistry Assessments of Mitophagy" (https://www.protocols.io/private/E5851436F8F111EF86990A58A9FEAC02).
RNA is extracted using the Monarch Total RNA Miniprep Kit (New England Bioscience) as per the manufacturers instructions and with inclusion of gDNA binding column and the optional on-column DNAse treatment.
RNA is eluted from the column in Amount30 µL of Nuclease free H2O
RNA is then quantified by 260nm absorbance using a NanoDrop One Spectrophotometer.
Reverse Transcription
Reverse Transcription
100ng of RNA is reverse transcribed in a Amount10 µL reaction with Concentration2.5 U/µl SuperScript IV reverse transcriptase, Concentration2.5 micromolar (µM) random hexamers, Concentration0.5 millimolar (mM) dNTPs, Concentration5 millimolar (mM) DTT and Concentration2 U/µl RNAseOUT.
A 100ng reverse transcription negative control reaction consisting of the same reaction mixture above but without reverse transcriptase is also conducted for each sample.
The reverse transcription and negative control reactions are thermocycled at:


TemperatureTimeCycles
25°C10minx1
50°C40min
80°C10min
Reverse Transcription Thermocycle

The cDNA product and reverse transcription negative control reactions are then diluted such that Amount100 ng of reverse transcribed RNA would be in a Amount120 µL final volume (i.e. add Amount10 µL reaction to Amount110 µL Nuclease Free H2O = Concentration0.83 ng/µl ).

RT-qPCR
RT-qPCR
3.33ng of cDNA (Amount4 µL of theConcentration0.83 ng/µl dilution) is used in each well of aAmount10 µL RT-qPCR reaction using Power Sybr Green PCR master mix, Concentration500 nanomolar (nM) gene-specific primer pairs and the QuantStudio 7 Flex Real-Time PCR System.
Each sample and gene target reaction is conducted with at least 2x technical replicates, with the same volume of reverse transcription negative control conducted alongside in a separate well for each gene target sample pair.

RT-qPCR Primer Pairs
PINK1_F: 5’- GTGGAACATCTCGGCAGGTT-3’
PINK1_R: 5’- CCTCTCTTGGATTTTCTGTAAGTGAC-3’
UBC_F: 5’-CTGGAAGATGGTCGTACCCTG-3’
UBC_R: 5’- GGTCTTGCCAGTGAGTGTCT-3’.  
Ct values are determined automatically using the Quantstudio Real-time PCR Software and relative PINK1 mRNA expression levels are calculated using the 2−ΔΔCt method with UBC as the house-keeping gene.
Protocol references
Soutar MPM, Melandri D, O'Callaghan B, Annuario E, Monaghan AE, Welsh NJ, D'Sa K, Guelfi S, Zhang D, Pittman A, Trabzuni D, Verboven AHA, Pan KS, Kia DA, Bictash M, Gandhi S, Houlden H, Cookson MR, Kasri NN, Wood NW, Singleton AB, Hardy J, Whiting PJ, Blauwendraat C, Whitworth AJ, Manzoni C, Ryten M, Lewis PA, Plun-Favreau H. Regulation of mitophagy by the NSL complex underlies genetic risk for Parkinson's disease at 16q11.2 and MAPT H1 loci. Brain. 2022 Dec 19;145(12):4349-4367. doi: 10.1093/brain/awac325. PMID: 36074904; PMCID: PMC9762952.