RNA Isolation from Embryonic Zebrafish and cDNA Synthesis for Gene Expression Analysis

Summary

The isolation of high quality, intact RNA is an essential step in many laboratory protocols. Here, we demonstrate RNA extraction from whole zebrafish embryos and cDNA synthesis for subsequent application in various experimental procedures including gene expression microarray analysis.

Abstract

Many important and complex laboratory procedures require an input of high quality, intact RNA. A degraded sample or the presence of impurities can lead to disastrous results in downstream experimental applications. It is therefore, of utmost importance to use solid techniques with numerous safeguards and quality control checks to ensure a superior sample. Herein, we detail a protocol to isolate total RNA from whole zebrafish embryos using a commercially available chemical denaturant and subsequent cleanup to remove traces of DNA and impurities using a commercial RNA isolation kit. As RNA is relatively unstable and easily prone to cleavage by RNAses, most protocols assay gene expression using a cDNA product that is directly synthesized from an RNA template. We detail a procedure to convert RNA into the more stable cDNA product using a commercially available kit. Throughout these procedures there are numerous quality control checks to ensure that the sample is not degraded or contaminated. The end product of these protocols is cDNA that is suitable for microarray analysis, RT-PCR or long-term storage.

Protocol

Part 1: Extraction of Total RNA using TRIzol Reagent

When working with RNA it is very important to work in an environment that is free of RNAses. Simple precautions such as having reserved pipettes for use only with RNA procedures and spraying the work area with a decontaminant reagent (e.g., RNAse Away) before beginning the procedure are very helpful.

1. To attain a sufficient amount of RNA pool 50 zebrafish embryos in a 1.5 ml microfuge tube and remove as much water as possible with a pipette.
2. Immediately add 250 μl of TRIzol reagent¹ to the microfuge tube containing the embryos. TRIzol reagent contains phenol ....

Discussion

The fragility of the RNA molecule is the most critical consideration that should be remembered throughout this protocol. Sterile equipment that is RNAse-free should always be used in an RNAse-free zone of the laboratory. It is helpful to reserve a part of the laboratory to be used for RNA procedures only. This area should be frequently sprayed with an RNAse removing product, such as RNAse Away. The RNA samples should always be handled with gloves and kept on ice to prevent degradation.

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Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Glycogen (5 mg/ml)	Ambion	9510
NanoDrop ND-100	Thermo Fisher Scientific, Inc.	ND-1000
Pellet Pestles with Microfuge Tube	VWR international	KT749520-0090
Phase Lock Gel Light, 1.5 ml	Eppendorf	2302800
Phenol, Tris-saturated	Roche Group	3117944001
RNAse Away	VWR international	17810-491
RNAse-Free DNase Set	Qiagen	79254
RNEasy Mini Kit	Qiagen	74104
SuperScript® First-Strand Synthesis System for RT-PCR	Invitrogen	11904-018
TRIzol	Invitrogen	15596-026