Cite as: Cold Spring Harb. Protoc.; 2009; doi:10.1101/pdb.prot5314

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Quantitative Real-Time RT-PCR (qRT-PCR) of Zebrafish Transcripts: Optimization of RNA Extraction, Quality Control Considerations, and Data Analysis

Chuan-Ching Lan1, Rongying Tang1, Ivone Un San Leong1, and Donald R. Love1,2,3

1 School of Biological Sciences, The University of Auckland, Auckland 1142, New Zealand
2 LabPLUS, Auckland City Hospital, Auckland 1148, New Zealand

3Corresponding author (DonaldL{at}adhb.govt.nz).


INTRODUCTION

The zebrafish (Danio rerio) has emerged as a popular model species. The rapid development of zebrafish embryos provides opportunities for investigation of genes essential for developmental processes, the human counterparts of which might be implicated in diseases. Understanding when and where genes are expressed can facilitate greater understanding of their function, and also allow the genes to be manipulated by gene knockdown in temporally and spatially specific manners. Quantitative real-time polymerase chain reaction (qRT-PCR) is widely applied in gene expression studies. This protocol presents techniques to optimize RNA isolation from zebrafish embryos; quality assessment and the use of multiple reference genes are also emphasized. The combined use of TRIzol extraction and column-based purification is strongly recommended, because the resulting RNA is of better quality than RNA isolated using either of those methods alone. The procedure can be performed in 2 d, with individual stages taking up to 15 h to complete.


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