Reference Genes across Nine Brain Areas of Wild Type and Prader-Willi Syndrome Mice: Assessing Differences in Igfbp7, Pcsk1, Nhlh2 and Nlgn3 Expression

Abstract

Prader–Willi syndrome (PWS) is a complex neurodevelopmental disorder caused by the deletion or inactivation of paternally expressed imprinted genes at the chromosomal region 15q11–q13. The PWS-critical region (PWScr) harbors tandemly repeated non-protein coding IPW-A exons hosting the intronic SNORD116 snoRNA gene array that is predominantly expressed in brain. Paternal deletion of PWScr is associated with key PWS symptoms in humans and growth retardation in mice (PWScr model). Dysregulation of the hypothalamic–pituitary axis (HPA) is thought to be causally involved in the PWS phenotype. Here we performed a comprehensive reverse transcription quantitative PCR (RT-qPCR) analysis across nine different brain regions of wild-type (WT) and PWScr mice to identify stably expressed reference genes. Four methods (Delta Ct, BestKeeper, Normfinder and Genorm) were applied to rank 11 selected reference gene candidates according to their expression stability. The resulting panel consists of the top three most stably expressed genes suitable for gene-expression profiling and comparative transcriptome analysis of WT and/or PWScr mouse brain regions. Using these reference genes, we revealed significant differences in the expression patterns of Igfbp7, Nlgn3 and three HPA associated genes: Pcsk1, Pcsk2 and Nhlh2 across investigated brain regions of wild-type and PWScr mice. Our results raise a reasonable doubt on the involvement of the Snord116 in posttranscriptional regulation of Nlgn3 and Nhlh2 genes. We provide a valuable tool for expression analysis of specific genes across different areas of the mouse brain and for comparative investigation of PWScr mouse models to discover and verify different regulatory pathways affecting this complex disorder.

Insulin secretion deficits in a Prader-Willi syndrome β-cell model are associated with a concerted downregulation of multiple endoplasmic reticulum chaperones

“…The latter include validation of (B) 10 down-regulated genes ( Derl3 , Mylip , Atp10a , Jph3 , Ndrg4 , Tap1 , Tap2 , Syt1 , Robo2 , Id4 ), as well as (C) for a set of 4 upregulated DEGs involved in the secretory pathway ( Chgb , Cacna1a , Tmem176a , Tmem176b ), with apparent upregulation for an exogenous human insulin ( Hs , Homo sapiens )-neomycin resistance ( Neo R) transgene in the INS-1 lines, although the latter is artifactual due to transgene silencing in line 16 (see S16A , S16I–S16K and S16M Fig ). (D,E) However, the RT-ddPCR analyses failed to validate (D) several candidate upregulated DEGs from the RNA-seq data ( Fig 4A ), or (E) candidates for downregulated DEGs based on data from PWS iPSC-derived neuronal and Snord116 -deficient mouse models [ 60 , 129 ]; likewise, another mouse study [ 130 ] did not confirm Pcsk1 . Statistical comparison by Welch’s t-test: *, P < 0.05; **, P < 0.005; ***, P < 0.0005.…”