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

Athanassios Fragoulis,Lucy Kathleen Reiss,Stefan Uhlig,Stephanie Fragoulis,Kristina Biller,Dennis Lex

doi:10.3390/ijms22157853

Athanassios Fragoulis, Lucy Kathleen Reiss + Show 4 more

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https://doi.org/10.3390/ijms22157853

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Abstract

qRT-PCR still remains the most widely used method for quantifying gene expression levels, although newer technologies such as next generation sequencing are becoming increasingly popular. A critical, yet often underappreciated, problem when analysing qRT-PCR data is the selection of suitable reference genes. This problem is compounded in situations where up to 25% of all genes may change (e.g., due to leukocyte invasion), as is typically the case in ARDS. Here, we examined 11 widely used reference genes for their suitability in commonly used models of acute lung injury (ALI): ventilator-induced lung injury (VILI), in vivo and ex vivo, lipopolysaccharide plus mechanical ventilation (MV), and hydrochloric acid plus MV. The stability of reference gene expression was determined using the NormFinder, BestKeeper, and geNorm algorithms. We then proceeded with the geNorm results because this is the only algorithm that provides the number of reference genes required to achieve normalisation. We chose interleukin-6 (Il-6) and C-X-C motif ligand 1 (Cxcl-1) as the genes of interest to analyse and demonstrate the impact of inappropriate normalisation. Reference gene stability differed between the ALI models and even within the subgroup of VILI models, no common reference gene index (RGI) could be determined. NormFinder, BestKeeper, and geNorm produced slightly different, but comparable results. Inappropriate normalisation of Il-6 and Cxcl1 gene expression resulted in significant misinterpretation in all four ALI settings. In conclusion, choosing an inappropriate normalisation strategy can introduce different kinds of bias such as gain or loss as well as under- or overestimation of effects, affecting the interpretation of gene expression data.

Highlights

Acute respiratory distress syndrome (ARDS) is a devastating inflammatory lung disease, affecting approximately 10% of all intensive care unit patients, whereof around40% perish in hospital [1]
To establish a procedure for normalisation based on reference genes, here, we examined the three most commonly used acute lung injury (ALI) models—each of them addressing different aetiologies of pulmonary inflammation—in a mouse intensive care unit (MICU)
To evaluate a valid and robust normalisation strategy for each experimental approach, we selected a set of 11 candidate reference genes (Table 1, reference genes/REF)

Summary

Introduction

Acute respiratory distress syndrome (ARDS) is a devastating inflammatory lung disease, affecting approximately 10% of all intensive care unit patients, whereof around40% perish in hospital [1]. Altered gene expression plays a critical role in ARDS, and it has been estimated that 10% to 25% of all genes may change their expression in this condition [4]. This impressive number illustrates the challenge of identifying suitable reference genes, which are indispensable as a yardstick to quantify changes in gene expression. To establish a procedure for normalisation based on reference genes, here, we examined the three most commonly used acute lung injury (ALI) models—each of them addressing different aetiologies of pulmonary inflammation—in a mouse intensive care unit (MICU)

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Conclusion