Apr 20, 2026

RT-qPCR (primary cells)

 Forked from Real-time qPCR
DOI
https://dx.doi.org/10.17504/protocols.io.261ger2b7l47/v1
  • 1Duke University;
  • 2KU Leuven;
  • 3Aligning 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.261ger2b7l47/v1
Protocol Citation: Shiyi Wang, Sarah van Veen 2026. RT-qPCR (primary cells). protocols.io https://dx.doi.org/10.17504/protocols.io.261ger2b7l47/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 09, 2025
Last Modified: April 20, 2026
Protocol  Integer ID: 117978
Keywords: ASAPCRN, qPCR, primary astrocytes, primary neurons, ATP13A4, atp13a4 expression in primary astrocyte, analyzing atp13a4 expression, details for atp13a4, atp13a4, reproducible assessment of gene expression, primary astrocyte, gene expression, housekeeping gene 18s for accurate quantification, primary cell, qpcr
Funders Acknowledgements:
Aligning Science Across Parkinson’s (ASAP) initiative
Grant ID: ASAP-020607
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Abstract
This protocol describes a qPCR-based method for analyzing Atp13a4 expression in primary astrocytes and neurons. Using a 96-well format, reactions are prepared with Fast SYBR Green Master Mix, specific forward and reverse primers, and nuclease-free water. Each sample is analyzed in technical replicates, with no-cDNA controls included to ensure specificity. Ct values are normalized to the housekeeping gene 18S for accurate quantification. Primer sequences and details for Atp13a4 and 18S are provided. This streamlined workflow enables reliable and reproducible assessment of gene expression and is accessible for further customization. 
Materials
  • Fast SYBR Green Master Mix (Cat# 43-856-16; Applied Biosystems)
  • Nuclease-free water
  • Forward (F) and reverse (R) primers (sequences below)
  • cDNA samples
  • 96-well qPCR plate
  • qPCR instrument

Forward (F) and reverse (R) primer sequences
  • Atp13a4: (F): 5’-GGCAGCCCACCTATACAAACTATATAT-3’ and (R): 5’-GAATGAAAAGACATACGCCCATCT-3’
  • 18S: (F) 5′-GCA​ATT​ATT​CCC​CAT​GAA​CG-3′ and (R) 5′-GGC​CTC​ACT​AAA​CCA​TCC​AA-3′.
Safety warnings
Follow institutional guidelines for the disposal of biological and chemical waste.
Plate cDNA samples on a 96-well qPCR plate.
For each well, prepare a reaction mix consisting of the following components:
  • 5 μL Fast SYBR Green Master Mix
  • 0.5 μL forward primer (10 μM)
  • 0.5 μL reverse primer (10 μM)
  • 4 μL cDNA sample (diluted to 12.5 ng/μL)
Add the prepared reaction mix to each well of the 96-well qPCR plate.
  • Include two to four technical replicates for each sample.
  • Add a no-cDNA control (negative control) containing all components except cDNA to check for contamination.
Record cycle threshold (Ct) values for each well.
Normalize Ct values to the housekeeping gene 18S.