RT-qPCR gene expression analysis in zebrafish: Preanalytical precautions and use of expressed repetitive elements for normalization

Methods Cell Biol. 2016;135:329-42. doi: 10.1016/bs.mcb.2016.02.002. Epub 2016 Mar 4.

Abstract

Gene expression analysis is increasingly important in many fields of biological research. Understanding patterns of expressed genes is assumed to provide insight into complex regulatory networks and can lead to the identification of genes relevant to specific biological processes, including disease. Among different techniques, reverse transcription quantitative polymerase chain reaction (RT-qPCR) is currently regarded as the gold standard for targeted quantification of RNA gene expression, especially because of its high sensitivity, specificity, accuracy, and precision, and also because of its practical simplicity and processing speed. However, different critical factors can influence the outcome of RT-qPCR studies, including isolation of RNA, reverse transcription to cDNA, and data analysis. These factors need to be addressed in order to obtain biologically meaningful results. In this chapter, we describe how RT-qPCR can be used in a reliable way to successfully study differential gene expression in zebrafish. Hereby, we especially focus on how expressed repetitive elements can be employed as reference targets in zebrafish RT-qPCR studies and how they can further improve the quality of the data.

Keywords: Expressed repeat elements; Normalization; Preanalytical steps; RNA extraction; RT-qPCR; Reference targets; Reverse transcription; Sample collection; Sample homogenization; Zebrafish.

MeSH terms

  • Animals
  • DNA, Complementary / genetics
  • Gene Expression Profiling / methods*
  • Gene Expression Regulation / genetics
  • RNA / biosynthesis
  • RNA / genetics
  • Real-Time Polymerase Chain Reaction / methods*
  • Repetitive Sequences, Nucleic Acid / genetics*
  • Zebrafish / genetics*

Substances

  • DNA, Complementary
  • RNA