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Padhi B.K.,HECSB | Rosales M.,HECSB | Pelletier G.,HECSB
NeuroToxicology | Year: 2015

Early life exposure to environmental chemicals can interfere with myelin formation in the developing brain, leading to neurological disorders. The Proteolipid Protein 1 (Plp1), Myelin Basic Protein (Mbp) and 2',3'- Cyclic Nucleotide 3'. Phosphodiesterase (Cnp) genes expressed in oligodendrocytes and involved in myelination processes can be useful biomarkers of potential developmental neurotoxicity. In an earlier study, we concluded that the reduction in the expression levels of Mbp splice variants in juvenile rat cerebellum following perinatal methylmercury (MeHg) exposure were compatible with an overall reduction of mature oligodendrocytes population. This observation prompted us to analyze the expression of Plp1 and Cnp in developing rat cerebellum to further confirm and investigate the toxic effects of MeHg on vulnerable oligodendrocytes. Splice variants of Plp1 in human and of Cnp in mouse are curated in NCBI RefSeq database, but not for rat. Lack of annotation of splice variants can pose significant challenge for the reliable quantification of gene expression levels in toxicological studies. Therefore, we applied a "comparative sequence analysis" approach, relying on annotated splice variants in human/mouse and on evolutionary conservation of intron-exon structures, to identify additional splice variants of Plp1 and Cnp in rat. Then, we confirmed their identity by nucleotide sequencing and characterized their temporal expression patterns during brain development by RT-PCR. The measurement of total transcripts and individual splice variants of Plp1 and Cnp in the cerebellum of MeHg-exposed rat pups revealed a relatively similar level of reduction in their expression levels. This study further confirms that perinatal exposure to MeHg can impact oligodendrocytes in pups. Based on these observations, we conclude that monitoring the expression of these oligodendrocyte-enriched genes can be useful to identify toxic chemicals affecting myelination. © 2015.


Padhi B.K.,HECSB | Singh M.,HECSB | Huang N.,HECSB | Pelletier G.,HECSB
Analytical Biochemistry | Year: 2016

Genomic DNA (gDNA) contamination of RNA samples can lead to inaccurate measurement of gene expression by reverse transcription quantitative real-time PCR (RT-qPCR). We describe an easily adoptable PCR-based method where gDNA contamination in RNA samples is assessed by comparing the amplification of intronic and exonic sequences from a housekeeping gene. Although this alternative assay was developed for rat RNA samples, it could be easily adapted to other species. As a proof of concept, we assessed the effects of detectable gDNA contamination levels on the expression of a few genes that illustrate the importance of RNA quality in acquiring reliable data. © 2015 Published by Elsevier Inc. All rights reserved.


Myelin sheaths surrounding axons are essential for saltatory conduction of nerve impulse in the central nervous system. A major protein constituent of myelin sheaths is produced by the myelin basic protein (Mbp) gene, whose expression in oligodendrocytes is conserved across vertebrates. In rat, five Mbp splice variants resulting from alternative splicing of exons 2, 5 and/or 6 are characterized. We developed a PCR-based strategy to quantify individual Mbp splice variants and characterized a sixth Mbp splice variant lacking only exon 5. This newly identified splice variant is predominantly expressed in developing rat brain and has orthologs in mouse and human. Many neurotoxic chemicals can perturb myelination and Mbp gene expression. Regulation of Mbp gene expression at the post-transcriptional level was assessed following perinatal exposure to neurotoxic methylmercury (2 mg/kg b.w./day). Similar reductions in total and individual Mbp splice variant mRNA levels suggest that methylmercury-induced perturbation in Mbp gene expression occurred as a consequence of decreased oligodendrocyte cell population in absence of a significant impact on its post-transcriptional regulation.


PubMed | HECSB
Type: | Journal: Analytical biochemistry | Year: 2015

Genomic DNA (gDNA) contamination of RNA samples can lead to inaccurate measurement of gene expression by reverse transcription quantitative real-time PCR (RT-qPCR). We describe an easily adoptable PCR-based method where gDNA contamination in RNA samples is assessed by comparing the amplification of intronic and exonic sequences from a housekeeping gene. Although this alternative assay was developed for rat RNA samples, it could be easily adapted to other species. As a proof of concept, we assessed the effects of detectable gDNA contamination levels on the expression of a few genes that illustrate the importance of RNA quality in acquiring reliable data.

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