Identification of reliable reference genes for gene expression studies in mouse models under microplastics stress.

Microplastics (MPs) and nanoplastics (NPs) have been deemed to be newly emerged contaminants interfering with various physiological processes closely related with gene expression alteration. Reverse transcriptase quantitative real-time polymerase chain reaction (RT-qPCR) serves as a powerful tool to assess gene expression, however highly dependent on a reliable reference gene. Therefore, it is necessary to identify stable reference genes for gene expression study under MP or NP stress. We constructed a mouse model postexposure to polypropylene microplastics (PP-MPs) to assess PP-MPs bioaccumulation in kidney, evaluate the kidney pathological changes, and then explore potential reference genes via RT-qPCR. Although the hematoxylin-eosin staining showed no obvious damage in kidney tissues, we observed significant PP-MPs accumulation in kidney using Raman spectra analysis supported by spectral multivariate analysis. The expression of 19 candidate reference genes were examined, including the commonly used ones of β-actin, glyceraldehyde 3-phosphate dehydrogenase (Gapdh), Cytochrome c oxidase subunit 4I1 (Cox4i), Histocompatibility 13 (H13) and ribosomal protein. Their expression stability and reliability were assessed by the combination of four algorithms including geNorm, NormFinder, BestKeeper and Delta Cq. The geNorm analysis revealed that the top three genes with the lowest variability were Cox4il, Rps9 and Gapdh, whereas NormFinder results ranked Rps3, Cox4il and Rps18 as the top three ones. Rpl15, Cox4i1 and Rps3 were the most reliable reference genes in BestKeeper results, and Delta Cq proposed Rps3 and Cox4il as the stable genes. The overall ranking indicated by GMR value gave the five most stable reference genes (Cox4i1, Rps3, Rps9, Rps18 and Gapdh). Three genes associated with different biochemical processes (Atp5f1, Crebbp and Dele1) were chosen to verify the characterized reference genes using the least stable gene as a control, exhibiting different expression profiles and implying the essentiality to select the reliable reference genes. Our results documented the expression fluctuations of acknowledged reference gene (Ubc) and proposed a set of reliable reference genes for future studies of gene expression profiles in MP treated mouse models.