Systematic analysis of candidate reference genes for gene expression analysis in hyperoxia-based mouse models of bronchopulmonary dysplasia

Abstract

Bronchopulmonary dysplasia is a chronic lung disease of preterm infants. Mouse models of hyperoxia-induced lung injury are often used to study pathogenesis and potential therapeutic approaches of BPD. Beside histological studies, gene expression analysis of lung tissue is typically used as experimental read out. RT-qPCR is the standard method for gene expression analysis, however, the accuracy of the quantitative data depends on the appropriate selection of reference genes. No data on validated reference genes for hyperoxia-induced neonatal lung injury in mice is available. In this study, 12 potential reference genes were systematically analyzed for their expression stability in lung tissue of neonatal mice exposed to room air or hyperoxia and healthy adult controls using published software algorithms. Analysis of gene expression data identified Hprt, Tbp and Hmbs as the most stable reference genes and proposed combinations of Hprt/Sdha or Hprt/Rpl13a as potential normalization factors. These reference genes and normalization factors were validated by comparing Il6 gene and protein expression and may facilitate accurate gene expression analysis in lung tissues of similar designed studies.

Selection and validation of suitable reference genes for gene expression studies in Callerya speciosa (Champ. ex Benth.) Schot under different experimental conditions

“…To date, some plants have been selected for research on RT-qPCR analysis, including Glycyrrhiza 27 , Nitraria tangutorum 20 , Fucus distichus 28 , Bromus sterilis 19 , Allium tuberosum 29 , and Suaeda glauca 30 . In addition, RT-qPCR is widely used in diagnosing diseases, such as preterm birth 31 , diabetic kidney disease 32 , Bronchopulmonary dysplasia 33 . 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 34,35 .…”

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

“…To date, some plants have been selected for research on RT-qPCR analysis, including Glycyrrhiza, Nitraria tangutorum, Fucus distichus, Bromus sterilis, Allium tuberosum, and Suaeda glauca . In addition, RT-qPCR is widely used in diagnosing diseases, such as preterm birth, diabetic kidney disease, and bronchopulmonary dysplasia . 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.…”

“…33 In addition, RT-qPCR is widely used in diagnosing diseases, such as preterm birth, 34 diabetic kidney disease, 35 and bronchopulmonary dysplasia. 36 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. 37,38 However, RT-qPCR requires a stable internal standard as a reference gene.…”

Selection and validation of suitable reference genes for gene expression studies in Callerya speciosa (Champ. ex Benth.) Schot under different experimental conditions

“…To date, some plants have been selected for research on RT-qPCR analysis, including Glycyrrhiza [27], Nitraria tangutorum [20], Fucus distichus [28], Bromus sterilis [19], Allium tuberosum [29], and Suaeda glauca [30]. In addition, RT-qPCR is widely used in diagnosing diseases, such as preterm birth [31], diabetic kidney disease [32], Bronchopulmonary dysplasia [33]. 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 [34,35].…”