Apr 15, 2026

RT-qPCR (cell lines)

DOI
https://dx.doi.org/10.17504/protocols.io.n92ldrq98g5b/v1
  • 1KU Leuven;
  • 2Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD 20815
Sarah van Veen
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DOI: https://dx.doi.org/10.17504/protocols.io.n92ldrq98g5b/v1
Protocol Citation: Sarah van Veen, Joris Van Asselberghs, Peter Vangheluwe 2026. RT-qPCR (cell lines). protocols.io https://dx.doi.org/10.17504/protocols.io.n92ldrq98g5b/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: January 12, 2025
Last Modified: April 15, 2026
Protocol  Integer ID: 118159
Keywords: ASAPCRN, RT-qPCR, ATP13A4, msatp13a4 mrna expression, lentiviral knockdown in c8, qpcr, following lentiviral knockdown, mrna expression, cell line, d1a cell, msatp13a4, use of rt, cell
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-000458
Fonds Wetenschappelijk Onderzoek (FWO)
Grant ID: G011424N
Abstract
This protocol describes the use of RT-qPCR to assess msAtp13a4 mRNA expression following lentiviral knockdown in C8-D1A cells. 
Materials
  • Cells with modulated ATP13A4 expression (e.g. C8-D1A cells with Atp13a4 knockdown)
  • NucleoSpin RNA Plus Kit (Macherey-Nagel, Cat# 740984.250)
  • High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems‱, Cat# 4374966)
  • SYBR Green qPCR Master Mix (Thermo Fisher or equivalent)
  • Nanodrop Spectrophotometer (Thermo Fisher)
  • Light Cycler (Roche)

Primers (custom synthesized):
  • msAtp13a4:
  • Forward (F): 5’-CCAGCATGCTTTACTCAATG-3’
  • Reverse (R): 5’-GAAGATGGATCCAATGAGAC-3’

  • msGapdh:
  • Forward (F): 5’-TGTGTCCGTCGTGGATCTGA-3’
  • Reverse (R): 5’-CCTGCTTCACCACCTTCTTGA-3’


Safety warnings
Follow institutional guidelines for the disposal of biological and chemical waste.
RNA extraction
Harvest 1.0 × 10⁶ cells from C8-D1A cultures.
Extract total RNA using the NucleoSpin RNA Plus Kit according to the manufacturer's protocol.

Measure RNA concentration and purity using a Nanodrop spectrophotometer.
cDNA synthesis
Reverse transcribe 1 μg of RNA into cDNA using the High-Capacity cDNA Reverse Transcription Kit, following the manufacturer's instructions.
qPCR
Prepare the qPCR reaction mix (per well):
  • 10 μl SYBR Green qPCR Master Mix
  • 1 μl forward primer (5 μM)
  • 1 μl reverse primer (5 μM)
  • 5 μl cDNA template (diluted 1:10 in nuclease-free water)
  • 3 μl nuclease-free water
Prepare reactions in duplicate for each target and reference gene.
Perform amplification using a Light Cycler with the following thermal cycling conditions:
  • Initial denaturation: 95 °C for 10 min
  • 50 cycles: 95 °C for 10 s, 55 °C for 30 s, 95 °C for 1 min, 55 °C for 1 min
Include a melting curve analysis from 55 °C to 95 °C.
Data analysis
Record the mean Cq values for all reactions.
Normalize target gene expression (Atp13a4) to the reference gene (Gapdh) using the ΔΔCq method.
Protocol references
van Veen S, Kourti A, Ausloos E, Van Asselberghs J, Van den Haute C, Baekelandt V, et al. ATP13A4 Upregulation Drives the Elevated Polyamine Transport System in the Breast Cancer Cell Line MCF7. Biomolecules. 2023;13(6).