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Reference Gene Selection for Gene Expression Analyses in Mouse Models of Acute Lung Injury.

Athanassios Fragoulis1, Kristina Biller2, Stephanie Fragoulis2

  • 1Department of Anatomy and Cell Biology, Uniklinik RWTH Aachen University, 52074 Aachen, Germany.

International Journal of Molecular Sciences
|August 7, 2021
PubMed
Summary

Selecting appropriate reference genes is crucial for accurate gene expression analysis using quantitative reverse transcription polymerase chain reaction (qRT-PCR). Inappropriate normalization in acute lung injury models leads to significant misinterpretation of results.

Keywords:
ALI mouse modelsIPLLPS-induced lung injuryVILIacid-induced lung injuryprimer validationqPCR normalisationreference gene establishment

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Area of Science:

  • Molecular Biology
  • Genomics
  • Respiratory Medicine

Background:

  • Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is a primary method for gene expression quantification.
  • Reference gene selection is critical but often overlooked in qRT-PCR data analysis, especially in complex conditions like acute lung injury (ALI).
  • Significant gene expression changes (up to 25%) can occur in ALI models, necessitating robust normalization strategies.

Purpose of the Study:

  • To evaluate the suitability of 11 commonly used reference genes in four distinct ALI models.
  • To assess reference gene stability using NormFinder, BestKeeper, and geNorm algorithms.
  • To demonstrate the impact of inappropriate normalization on the interpretation of key genes (interleukin-6 and C-X-C motif ligand 1) in ALI.

Main Methods:

  • Tested 11 reference genes across ventilator-induced lung injury (VILI), lipopolysaccharide plus mechanical ventilation, and hydrochloric acid plus mechanical ventilation ALI models.
  • Employed NormFinder, BestKeeper, and geNorm algorithms to determine reference gene expression stability.
  • Utilized geNorm for its ability to specify the optimal number of reference genes for normalization.
  • Analyzed interleukin-6 (Il-6) and C-X-C motif ligand 1 (Cxcl-1) expression to illustrate normalization effects.

Main Results:

  • Reference gene stability varied significantly across different ALI models and even within VILI subgroups.
  • No universal reference gene index (RGI) was identified across all tested ALI models.
  • NormFinder, BestKeeper, and geNorm provided comparable, though slightly different, stability assessments.
  • Inappropriate normalization of Il-6 and Cxcl-1 led to substantial misinterpretations in all four ALI settings.

Conclusions:

  • Reference gene selection is model-dependent and critical for accurate gene expression analysis in ALI.
  • Inappropriate normalization strategies can introduce bias, leading to under- or overestimation of biological effects.
  • Careful validation of reference genes is essential to ensure reliable interpretation of qRT-PCR data in respiratory research.