Validation of Housekeeping Genes as Internal Controls for the Study of the Effects of Microcystin-LR in Zebrafish by Real-Time PCR

Zebrafish. 2018 Oct;15(5):454-459. doi: 10.1089/zeb.2018.1598. Epub 2018 Jul 25.

Abstract

Microcystin-leucine arginine (MC-LR) is a natural toxin produced by cyanobacterial blooms that causes severe liver damage in fish. It is crucial to investigate if housekeeping genes are affected by MC-LR in zebrafish, to permit the adequate evaluation of gene expression by RT-qPCR. We evaluated the gene expression stability (M value) and regulation by chemical treatment (using E-Ct and E-ΔCt) to validate the use of eight housekeeping genes in fish exposed to 0, 0.31, and 6.10 μg L-1 MC-LR for 24 h. We suggest the use of the combination of β-actin1, b2m, and arnt2 in the liver and β-actin1, 18S rRNA, and arnt2 in gills as housekeeping genes. The evaluation of gene regulation following MC-LR exposure denoted a strong repression of 18S rRNA (17- and 10-fold decrease) and tbp (10- and 2-fold decrease) and induction of ef-1α (8- and 14-fold increase) in the liver of zebrafish exposed to 0.31 and 6.10 μg L-1 MC-LR, respectively. This is the first study showing that housekeeping genes commonly used in gene expression could be affected in the liver by environmentally relevant concentrations of MC-LR. The study validates adequate housekeeping genes that could be used in toxicological studies with MC-LR in zebrafish.

Keywords: Danio rerio; MC-LR; RT-qPCR; reference gene.

Publication types

  • Research Support, Non-U.S. Gov't
  • Validation Study

MeSH terms

  • Animals
  • Enzyme Inhibitors / pharmacology*
  • Gene Expression Regulation / drug effects*
  • Genes, Essential*
  • Liver / drug effects
  • Liver / metabolism*
  • Marine Toxins
  • Microcystins / pharmacology*
  • Real-Time Polymerase Chain Reaction / methods*
  • Real-Time Polymerase Chain Reaction / standards
  • Reference Standards
  • Zebrafish

Substances

  • Enzyme Inhibitors
  • Marine Toxins
  • Microcystins
  • cyanoginosin LR