May 08, 2025

Public workspaceGene expression analysis

DOI
https://dx.doi.org/10.17504/protocols.io.yxmvmywebv3p/v1
  • Lucas Blasco-Agell1,2,
  • Meritxell Pons-Espinal1,2,
  • Jara Montero-Muñoz1,2,
  • Angel Raya1,2,
  • Antonella Consiglio1,2,
  • Miquel Vila3
  • 1Department of Pathology and Experimental Therapeutics, Bellvitge University Hospital- IDIBELL, 08908 Hospitalet de Llobregat, Spain;
  • 2Institute of Biomedicine of the University of Barcelona (IBUB), Carrer Baldiri Reixac 15-21, Barcelona 08028, Spain;
  • 3VHIR-CIBERNED-ASAP
  • Vilalab Public
Miquel Vila
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DOI: https://dx.doi.org/10.17504/protocols.io.yxmvmywebv3p/v1
Protocol Citation: Lucas Blasco-Agell, Meritxell Pons-Espinal, Jara Montero-Muñoz, Angel Raya, Antonella Consiglio, Miquel Vila 2025. Gene expression analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvmywebv3p/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: April 17, 2025
Last Modified: May 08, 2025
Protocol Integer ID: 130936
Keywords: human microglia cell, neuromelanin, gene expression, neurotoxic factor, gene expression analysis
Funders Acknowledgements:
ASAP
Grant ID: ASAP-020505
Abstract
To evaluate whether human microglia cells (hMG) can be activated by neuromelanin (NM), hMG were cultured with and without the presence of NM for 24 h and then harvested to measure gene expression of selected pro-inflammatory and neurotoxic factors.
Troubleshooting
RNA is extracted using a RNeasy‱ Mini kit (Qiagen) according to manufacturer’s instructions and quantified with a NanoDrop one spectrophotometer (Thermo Fisher Scientific).
Reverse transcription into complementary cDNA is performed with the Superscript III First-Strand Synthesis System (Invitrogen‱) to a final concentration of 2 ng/μL.
Gene expression is measured by real time quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR).
8 ng of cDNA are loaded in every well of a MicroAmp‱ Optical 384-well Reaction Plate (Applied Biosystems‱) with 10 nM of forward and reverse primers (Life Technologies) and PowerUp‱ SYBR‱ Green Master Mix (Applied Biosystems‱).
Plate is run in a 7900HT Fast Real-Time PCR System with 384-well Block Module (Applied Biosystems‱) following a standard cycling mode.
Cycling values are normalized to the housekeeping gene β-Actin and the relative fold gene expression of samples was calculated using the 2-ΔΔCt method.