Feb 03, 2025

Public workspaceQuantitative RT-PCR

DOI
dx.doi.org/10.17504/protocols.io.kqdg3q6zzv25/v1
  • 1Institut Imagine
  • Team Deleidi
Federico Bertoli
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DOI: dx.doi.org/10.17504/protocols.io.kqdg3q6zzv25/v1
Protocol Citation: Maria Jose Perez J. 2025. Quantitative RT-PCR. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg3q6zzv25/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: November 26, 2024
Last Modified: February 03, 2025
Protocol Integer ID: 112820
Funders Acknowledgements:
ASAP
Abstract
Quantitative RT-PCR
Quantitative RT-PCR
Quantitative RT-PCR
Isolate mRNA from iPSC-derived neurons, astrocytes, and microglia using the RNeasy Mini Kit (Cat. number 74106, QIAGEN, Germany) following the manufacturer’s instructions.
Perform reverse transcription of the isolated RNA using the QuantiTect Reverse Transcription Kit (Cat. number 205313, QIAGEN) to synthesize cDNA.
Set up quantitative PCR reactions using SYBR Green PCR Master Mix (Cat. number 204145, QIAGEN).
Conduct qPCR in a Viia 7 Real-Time PCR System (Applied Biosystems, CA, USA).
Normalize the expression level of each target gene to the housekeeping genes ribosomal protein large P0 (RPLP0) or actin to account for RNA input variability.
Calculate relative gene expression levels using the 2−ΔΔCT method.
Determine fold changes in gene expression based on the calculated relative expression values.
Refer to Supplementary Table 2 of the study for the primer sequences used in the reactions.