Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

Summary

Microarray technology allows quantitative measurement and gene expression profiling of transcripts on a genome-wide basis. Therefore, this protocol provides an optimized technical procedure in a two-color custom made bovine array using Day 7 bovine embryos to demonstrate the feasibility of using low amount of total RNA.

Abstract

Early embryonic loss is a large contributor to infertility in cattle. Moreover, bovine becomes an interesting model to study human preimplantation embryo development due to their similar developmental process. Although genetic factors are known to affect early embryonic development, the discovery of such factors has been a serious challenge. Microarray technology allows quantitative measurement and gene expression profiling of transcript levels on a genome-wide basis. One of the main decisions that have to be made when planning a microarray experiment is whether to use a one- or two-color approach. Two-color design increases technical replication, minimizes variability, improves sensitivity and accuracy as well as allows having loop designs, defining the common reference samples. Although microarray is a powerful biological tool, there are potential pitfalls that can attenuate its power. Hence, in this technical paper we demonstrate an optimized protocol for RNA extraction, amplification, labeling, hybridization of the labeled amplified RNA to the array, array scanning and data analysis using the two-color analysis strategy.

Introduction

Early embryonic loss in high-producing dairy cows is one of the main challenges in the dairy industry^1,2. Bovine has become an interesting model to study human preimplantation embryo development due to their similar developmental process^3,4. However, more research is required to have better understanding about genes involved in bovine early embryonic development.

After twenty years since the first microarray technology developed in 1995⁵, the development of more sophisticated probe fabrication technol....

Protocol

The animal part of this study was conducted at the Metabolic Research Unit of the University of Alberta, Edmonton, Canada, with all animal experimental procedures approved (Protocol # AUP00000131) by the University of Alberta Animal Care and Use Committee, and animals cared for according to the Canadian Council of Animal Care guidelines (1993).

1. Embryo Production, Isolation of Total RNA and DNase Treatment

1. For animal experimental protocols and embryo collection refer in our previous publications^{17, 18}.
2. Pool and snap freeze similar stage embryos from each cow in liquid nitrogen an....

Results

A representative result of total RNA and amplified aRNA from Day 7 bovine embryos is shown in Figure 3 and summarized in Table 1.

RNA integrity and profile can be assessed after RNA extraction. Quality assessment of RNA could be done by bioanalyzer instrument (Figure 3A) and only those samples with RIN value higher than 7.0 are qualified to be used for amplification (Tab.......

Discussion

The first problem to perform microarray analysis using Day 7 bovine embryos is not getting sufficient amounts of high quality RNA for studying gene expression. Traditional phenol/chloroform RNA extraction and ethanol precipitation method is not recommended for Day 7 embryos, resulting in low yield and possible leftover phenol inhibiting RNA amplification reaction. Instead, a standard column-based method is better to isolate total RNA and then elute the RNA with minimum elution buffer to increase the concentration. An ave.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
PicoPure RNA Isolation Kit	Applied Biosystems	KIT0204
RNase-Free DNase Set (50)	Qiagen	79254
Agilent RNA 6000 Pico Kit	Agilent Technologies	5067-1513
Arcturus RiboAmp HS PLUS Kit	Applied Biosystems	KIT0505
2100 Bioanalyzer Instruments	Agilent Technologies	G2940CA
RNA Screen Tape	Agilent Technologies	5067-5576
ULS Fluorescent Labeling Kit	Kreatech Diagnostics	EA-021
Custom Gene Expression Microarrays	Agilent Technologies	G2514F
Agilent Gene Expression wash buffer 1	Agilent Technologies	Part #5188-5325
Agilent Gene Expression wash buffer 2	Agilent Technologies	Part #5188-5326
2x Hi-RPM Hybridization buffer	Agilent Technologies	Part #5190-0403
25x Fragment buffer	Agilent Technologies	Part #5185-5974
10x GE Blocking Agent	Agilent Technologies	Part #5188-5281
Stabilization and drying solution	Agilent Technologies	Part #5185-5979
Gasket slides enabled by Agilent SureHyb techonolgy	Agilent Technologies	G2524-60012	Pack of 20 gasket slides, 4 microarrays/slide
Two-Color RNA Spike-In Kit	Agilent Technologies	Cat# 5188-5279
GenePix 4000B array scanner	Molecular Devices	GENEPIX 4000B-U
Ozone Free Box	BioTray	OFB_100x200
GAL file	Agilent Technologies	-