Abstract
Choroid plexus (ChP), a vascularized secretory epithelium located in all brain ventricles, plays critical roles in development, homeostasis and brain repair. Reverse transcription quantitative real-time PCR (RT-qPCR) is a popular and useful technique for measuring gene expression changes and also widely used in ChP studies. However, the reliability of RT-qPCR data is strongly dependent on the choice of reference genes, which are supposed to be stable across all samples. In this study, we validated the expression of 12 well established housekeeping genes in ChP in 2 independent experimental paradigms by using popular stability testing algorithms: BestKeeper, DeltaCq, geNorm and NormFinder. Rer1 and Rpl13a were identified as the most stable genes throughout mouse ChP development, while Hprt1 and Rpl27 were the most stable genes across conditions in a mouse sensory deprivation experiment. In addition, Rpl13a, Rpl27 and Tbp were mutually among the top five most stable genes in both experiments. Normalisation of Ttr and Otx2 expression levels using different housekeeping gene combinations demonstrated the profound effect of reference gene choice on target gene expression. Our study emphasized the importance of validating and selecting stable housekeeping genes under specific experimental conditions.
Highlights
Choroid plexus (ChP) is a highly vascularized tissue located within the four brain ventricles
It has been demonstrated that ChP has the intrinsic ability to sense external changes associated with central nervous system (CNS) a ctivity[7,8] and it is well known that ChP produces and releases Otx[2], an essential factor implicated in both the onset and the closure of visual critical period[9]
The 12 candidate reference genes used in this study were selected based on their distinct cellular function and on their extensive use in neuroscience researches[18,22,23,30,31]
Summary
Choroid plexus (ChP) is a highly vascularized tissue located within the four brain ventricles. Changes in ChP-CSF structure and function have been linked to neurodegenerative diseases such as Alzheimer’s d isease[10], to neurodevelopmental disorders[11] such as autism-spectrum disorder[12] and schizophrenia[13] and to neuroimmune disease such as multiple sclerosis[14]. This increasing interest in ChP parallels the need for gene expression studies of ChP in different experimental contexts. Gene name (MGI) Actin, beta ATP synthase peripheral stalk-membrane subunit b Beta-2 microglobulin Glyceraldehyde-3-phosphate dehydrogenase Hypoxanthine guanine phosphoribosyl transferase Phosphoglycerate kinase 1 Retention in endoplasmic reticulum sorting receptor 1 Ribosomal protein L13A Ribosomal protein L27 Succinate dehydrogenase complex, subunit A, flavoprotein (Fp) TATA box binding protein Ubiquitin C Transthyretin Orthodenticle homeobox 2
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