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.

BACKGROUND:Androgens play an important role for the development of male fertility and gained interest as growth and survival factors for certain types of cancer. Androgens act via the androgen receptor (AR/Ar), which is involved in various cell biological processes such as sex differentiation. To study the functional mechanisms of androgen action, cell culture systems and AR-transfected cell lines are needed. Transfection of AR into cell lines and subsequent gene expression analysis after androgen treatment is well established to investigate the molecular biology of target cells. However, it remains unclear how the transfection with AR itself can modulate the gene expression even without androgen stimulation. Therefore, we transfected Ar-deficient rat Sertoli cells 93RS2 by electroporation using a full length human AR.RESULTS:Transfection success was confirmed by Western Blotting, immunofluorescence and RT-PCR. AR transfection-related gene expression alterations were detected with microarray-based genome-wide expression profiling of transfected and non-transfected 93RS2 cells without androgen stimulation. Microarray analysis revealed 672 differentially regulated genes with 200 up- and 472 down-regulated genes. These genes could be assigned to four major biological categories (development, hormone response, immune response and metabolism). Microarray results were confirmed by quantitative RT-PCR analysis for 22 candidate genes.CONCLUSION:We conclude from our data, that the transfection of Ar-deficient Sertoli cells with AR has a measurable effect on gene expression even without androgen stimulation and cause Sertoli cell damage. Studies using AR-transfected cells, subsequently stimulated, should consider alterations in AR-dependent gene expression as off-target effects of the AR transfection itself.