Sample Preparation and Analysis of RNASeq-based Gene Expression Data from Zebrafish

Summary

This protocol presents an approach for whole transcriptome analysis from zebrafish embryos, larvae, or sorted cells. We include isolation of RNA, pathway analysis of RNASeq data, and qRT-PCR-based validation of gene expression changes.

Abstract

The analysis of global gene expression changes is a valuable tool for identifying novel pathways underlying observed phenotypes. The zebrafish is an excellent model for rapid assessment of whole transcriptome from whole animal or individual cell populations due to the ease of isolation of RNA from large numbers of animals. Here a protocol for global gene expression analysis in zebrafish embryos using RNA sequencing (RNASeq) is presented. We describe preparation of RNA from whole embryos or from cell populations obtained using cell sorting in transgenic animals. We also describe an approach for analysis of RNASeq data to identify enriched pathways and Gene Ontology (GO) terms in global gene expression data sets. Finally, we provide a protocol for validation of gene expression changes using quantitative reverse transcriptase PCR (qRT-PCR). These protocols can be used for comparative analysis of control and experimental sets of zebrafish to identify novel gene expression changes, and provide molecular insight into phenotypes of interest.

Introduction

Comparative analysis of global gene expression is a valuable tool to identify novel genes contributing to observed phenotypes. Such analyses typically rely on quantitative assessment of transcript abundance compared between experimental and control samples. Targeted approaches, such as qRT-PCR are relatively rapid and accurate for investigation of single gene expression changes. RNA sequencing (RNASeq) offers a broad, hypothesis-free approach to identify significant changes in gene expression between samples, making it now the standard for such investigations across experimental systems.

Zebrafish have emerged as a prominent model across ma....

Protocol

All animal protocols outlined below are in accordance with and approved by the University of Maryland Institutional Animal Care and Use Committee (IACUC).

1. Embryo Preparation

1. Generating embryos through natural mating
   1. Culture embryos to 3 months of age, reproductive maturity^5,8.
   2. Segregate adult male and female fish from the desired strain into divided mating tanks on the evening before embryo collection, and add 2 males and 3 females to each tank.
      NOTE: Use of the transgenic insulin2a:mCherry fluorescent reporter st....

Results

Sorting of Differentially Expressed Genes:

To identify differentially expressed genes in the larval stage of zebrafish models of Alström Syndrome and Bardet-Biedl Syndrome (BBS), we targeted either alms1 or bbs1 transcripts by injecting previously validated splice-blocking MOs into wild-type zebrafish embryos^16,17. At 5 days post fertilization (dpf), two r.......

Discussion

The approach described in this protocol offers a relatively rapid and cost-effective strategy for transcriptome-level analysis of whole animals or specific sorted cell populations. The zebrafish provides an advantageous model for this type of study because of the ease and rapidity in generating large amounts of starting material, the ease of implementing genetic or environmental experimental conditions, and the availability of a large spectrum of transgenic reporter lines allowing for isolation of cell-type specific and .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Commercial Reagents
TriZol	Thermo Scientific	15596026	lysis reagent
TrypLE	Gibco	12604013	dissociation buffer 1
FACSMax	Genlantis	T200100	dissociation buffer 2
DEPC-treated water	Sigma	95284
FirstStrand cDNA conversion	Thermo Scientific	K1621	cDNA conversion kit
2X SYBR Green Master Mix	Roche	4707516001	qRT-PCR Master Mix
FACS buffer	Fisher Scientific	50-105-9042
chloroform	Sigma Aldrich	288306
sodium acetate	Sigma Aldrich	S2889
Name	Company	Catalog Number	Comments
Zebrafish Strains
Tuebingen	ZIRC	ZL57
ins2a:mCherry	ZIRC	ZL1483
Name	Company	Catalog Number	Comments
Equipment
40 micron cell strainer	Sigma	CLS431750
FACS tube	BD Falcon	352063
hemocytometer	Sigma	Z359629
Dissecting Microscope	Zeiss
Inverted Microscope	Zeiss
Nanodrop	Thermo Scientific
Illumina HiSeq	Illumina
LightCycler 480	Roche
Mating tanks 1.0L Crossing Tank Set	Aquaneering	ZHCT100
FACS tube 5 mL polypropylene tube	BD Falcon	352063
Name	Company	Catalog Number	Comments
Software
Excel	Microsoft
Consensus Path DB	http://cpdb.molgen.mpg.de/
GO Enrichment Analysis	http://geneontology.org/page/go-enrichment-analysis