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

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.

Physiological Mineralization during In Vitro Osteogenesis in a Biomimetic Spheroid Culture Model

“…Amplification efficiencies were determined using LinRegPCR 2016 software (Academic Medical Centre, Amsterdam, The Netherlands) and relative gene expression was calculated after normalization to previously defined reference genes (mHprt, mSdha) according to a geNorm study, which was conducted prior the gene expression study. Qbase+ 3.3 software (Biogazelle, Gent, Belgium) was used to calculate relative changes in target gene expression according to an efficiency-corrected ΔΔCq method [ 41 , 42 ].…”

A juvenile murine model with chronic lung inflammation induced by repeated intratracheal instillation of lipopolysaccharides: a versatile and replicable model

“…Previous studies (19,20) showed that intratracheal instillation 2 mg/kg of LPS can cause acute lung injury in adult C57BL/6 mice. Acute inflammation peaks 2 days after instillation, and then significantly alleviated 7 days after instillation (19).…”

Selection and Validation of Suitable Reference Genes for Gene Expression Studies in Callerya speciosa (Champ. ex Benth.) Schot under Different Experimental Conditions

“…Due to its rapidity and accuracy in informing on active coronavirus ( CoV ) infection, RT-qPCR has become the assay of choice for COVID-19 diagnosis. , However, RT-qPCR requires a stable internal standard as a reference gene. Previous studies reported that the expression profiles of reference genes might be unstable under different species, tissues, developmental stages, and abiotic stress. ,, An unstable reference gene will cause misleading and even contradictory results. , Therefore, it is always necessary to validate the optimal reference genes prior to their applications.…”

“…Previous studies reported that the expression pro les of reference genes might be unstable under different species, tissues, developmental stages, and abiotic stress [9,18,36]. And an unstable reference gene will cause misleading and even contradictory results [37,38]. Therefore, it's always necessary to validate the optimal reference genes prior to their applications.…”

“…9,18,39 An unstable reference gene will cause misleading and even contradictory results. 40,41 Therefore, it is always necessary to validate the optimal reference genes prior to their applications.…”