Reference genes for the developing mouse lung under consideration of biological, technical and experimental confounders

Abstract

For gene expression analysis, the raw data obtained from RT-qPCR are preferably normalized to reference genes, which should be constantly expressed regardless of experimental conditions. Selection of reference genes is particularly challenging for the developing lung because of the complex transcriptional and epigenetic regulation of genes during organ maturation and injury repair. To date, there are only limited experimental data addressing reliable reference genes for this biological circumstance. In this study, we evaluated reference genes for the lung in neonatal C57BL/6 mice under consideration of biological, technical and experimental conditions. For that, we thoroughly selected candidates from commonly used reference genes side-by-side with novel ones by analyzing publicly available microarray datasets. We performed RT-qPCR of the selected candidate genes and analyzed their expression variability using GeNorm and Normfinder. Cell-specific expression of the candidate genes was analyzed using our own single-cell RNA-sequencing data from the developing mouse lung. Depending on the investigated conditions, i.e., developmental stages, sex, RNA quality, experimental condition (hyperoxia) and cell types, distinct candidate genes demonstrated stable expression confirming their eligibility as reliable reference genes. Our results provide valuable information for the selection of proper reference genes in studies investigating the neonatal mouse lung.

Figure 1

Profile of reference gene expression time in the developing lung by single-cell RNA sequencing. Using a recently published single-cell sequencing atlas of the developing mouse lung, gene expression of the different candidate reference genes in broadly categorized cell types (epithelial, endothelial and mesenchymal cells) was analyzed over time. The average expression in each cell type at each timepoint is plotted in a dotplot. The size of the circle indicates the fraction of cells with expression levels above the limit of detection and the intensity of the color of the circles indicates the average expression level in each cell. Larger circles indicate a greater fraction of cells with detectable expression and darker circles indicates greater average expression.

Figure 2

Marker gene expression was profiled in specific cell types by single-cell RNA sequencing. From the single-cell sequencing atlas of mouse lung development, expression across the different cell types was averaged from mouse lungs collected at P0, P3, P5, P7 and P14 and plotted on a heatmap. Darker colors indicate greater expression.

Figure 3

Reference genes with low expression variability (RT-qPCR). Black, bold: Genes with high expression stability identified by both GeNorm and Normfinder. Colored: Genes selected for low expression variability by GeNorm (green) oder Normfinder (red). Pcsk7 (blue) was identified by GeNorm in the hyperoxia condition and by Normfinder in the physiologic condition.

Figure 4

The reliable reference genes with regard to the analytical approaches. 16 out of 23 reference gene candidates that were identified in at least one analytical approach (colored bars) as a reliable reference gene are presented. Maea was most commonly identified as stably expressed gene among the different analytical approaches. For detailed information about experimental conditions of the analytical approaches see the text.