Sep 14, 2022

Public workspaceGene expression analysis by quantitative Real-Time PCR (qPCR)

Nature Communications
DOI
https://dx.doi.org/10.17504/protocols.io.ewov1oyz2lr2/v1
  • Miquel Vila1
  • 1Vall d'Hebron Research Institute
  • Vilalab Public
  • Nuria
Núria Peñuelas
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DOI: https://dx.doi.org/10.17504/protocols.io.ewov1oyz2lr2/v1
External link: https://doi.org/10.1038/s41467-024-53168-7
Protocol Citation: Miquel Vila 2022. Gene expression analysis by quantitative Real-Time PCR (qPCR). protocols.io https://dx.doi.org/10.17504/protocols.io.ewov1oyz2lr2/v1
Manuscript citation:
Laguna, A., Peñuelas, N., Gonzalez-Sepulveda, M. et al. Modelling human neuronal catecholaminergic pigmentation in rodents recapitulates age-related neurodegenerative deficits. Nat Commun 15, 8819 (2024). https://doi.org/10.1038/s41467-024-53168-7
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: September 13, 2022
Last Modified: September 14, 2022
Protocol Integer ID: 69945
Keywords: gene expression analysis, qpcr, time pcr
Abstract
Quantitative Real-Time PCR (qPCR)
Troubleshooting
qPCR plate reagents
Option 1: TaqMan qPCR
Prepare Taqman Master mix (ul per well):
  • 5ul: Taqman Gene Expression Master Mix (Applied Biosystems, # 4369016)
  • 0.5ul Taqman Gene Expression Assays

Add 5.5ul of Taqman Master mix to each well with an automatic pipette (Multipette E3, Eppendorf)
Add 4.5ul of cDNA per well (10ng cDNA) in technical triplicates to each well of LightCycler 480 Multiwell 384-plate (#4729749001, Roche Diagnostics)
Option 2: SYBR qPCR
Prepare SYBR Master mix (ul per well):
  • 5ul PowerUp SYBR Green Master Mix (#A25776, Applied Biosystem-ThermoFisher)
  • 1ul Primers (Forward and Reverse, diluted in Nuclease-free water at 5µM)

Add 6ul of SYBR Master mix to each well with an automatic pipette (Multipette E3, Eppendorf)
Add 4ul of cDNA per well (10ng cDNA) in technical triplicates to each well of LightCycler 480 Multiwell 384-plate (#4729749001, Roche Diagnostics)
Thermocycling
Perform PCR using the following cycling conditions in a LightCycler® 480 System (Roche):
  • 50˚C for 2min
  • 95˚C for 2min
  • 95˚C for 15s + 60˚C for 1min (x40cycles)
Data an
Using the LightCycler Software obtain the threshold cycles (CT) signals from each samples.
Perform ΔΔCT-method to analyze the expression data using the endogenous control genes and the reference experimental group.