Research supported by Alberta Livestock and Meat Agency, Alberta Innovates - BioSolutions, Alberta Milk, and Livestock Research Branch, Alberta Agriculture and Forestry.

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
<ol>
	<li>For animal experimental protocols and embryo collection refer in our previous publications<sup ...

<ol>
	<li>Royal, M. D., Smith, R. F., Friggens, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fertility+in+dairy+cows:+bridging+the+gaps.">Fertility in dairy cows: bridging the gaps.</a> Animal. 2 (08), 1101-1103 (2008).</li><li>Diskin, M. G., Murphy, J. J., Sreenan, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryo+survival+in+dairy+cows+managed+under+pastoral+conditions.">Embryo survival in dairy cows managed under pastoral conditions.</a> Anim. Reprod. Sci. 96 (3-4), 297-311 (2006).</li><li>Wrenzycki, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+culture+system+and+protein+supplementation+on+mRNA+expression+in+pre-implantation+bovine+embryos.">Effects of culture system and protein supplementation on mRNA expression in pre-implantation bovine embryos.</a> Hum. Reprod. 16 (5), 893-901 (2001).</li><li>Menezo, Y. J., Herubel, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+and+bovine+models+for+human+IVF.">Mouse and bovine models for human IVF.</a> Reprod. Biomed. Online. 4 (2), 170-175 (2002).</li><li>Schena, M., Shalon, D., Davis, R. W., Brown, P. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+monitoring+of+gene+expression+patterns+with+a+complementary+DNA+microarray.">Quantitative monitoring of gene expression patterns with a complementary DNA microarray.</a> Science. 270 (5235), 467-470 (1995).</li><li>Patterson, T. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Performance+comparison+of+one-color+and+two-color+platforms+within+the+MicroArray+Quality+Control+(MAQC)+project.">Performance comparison of one-color and two-color platforms within the MicroArray Quality Control (MAQC) project.</a> Nat. Biotechnol. 24 (9), 1140-1150 (2006).</li><li>Rhodes, D. R., Chinnaiyan, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrative+analysis+of+the+cancer+transcriptome.">Integrative analysis of the cancer transcriptome.</a> Nat. Genetics. 37, 31-37 (2005).</li><li>Robert, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combining+resources+to+obtain+a+comprehensive+survey+of+the+bovine+embryo+transcriptome+through+deep+sequencing+and+microarrays.">Combining resources to obtain a comprehensive survey of the bovine embryo transcriptome through deep sequencing and microarrays.</a> Mol. Reprod. 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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diets+enriched+in+unsaturated+fatty+acids+enhance+early+embryonic+development+in+lactating+Holstein+cows.">Diets enriched in unsaturated fatty acids enhance early embryonic development in lactating Holstein cows.</a> Theriogenology. 68 (7), 949-957 (2007).</li><li>. Agilent 2100 Bioanalyzer User's Guide Available from: <a target="_blank" href="https://www.agilent.com/cs/library/usermanuals/Public/G2946-90004_Vespucci_UG_eBook_(NoSecPack)">https://www.agilent.com/cs/library/usermanuals/Public/G2946-90004_Vespucci_UG_eBook_(NoSecPack)</a> (2016)</li><li>Kerr, K. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extended+analysis+of+benchmark+datasets+for+Agilent+two-color+microarray.">Extended analysis of benchmark datasets for Agilent two-color microarray.</a> BMC Bioinformatics. 8, 371 (2007).</li><li>Zhu, Q., Miecznikowski, J. C., Halfon, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+wholly+defined+Agilent+microarray+spike-in+dataset.">A wholly defined Agilent microarray spike-in dataset.</a> Bioinformatics. 27 (9), 1284-1289 (2011).</li><li>Vallee, M., Gravel, C., Palin, M. F., Reghenas, H., Stothard, P., Wishart, D. S., Sirard, M. 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D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PANTHER:+a+library+of+protein+families+and+subfamilies+indexed+by+function.">PANTHER: a library of protein families and subfamilies indexed by function.</a> Genome Res. 13 (9), 2129-2141 (2003).</li><li>Mi, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+PANTHER+database+of+protein+families,+subfamilies,+functions+and+pathways.">The PANTHER database of protein families, subfamilies, functions and pathways.</a> Nucleic Acids Res. 33, 284-288 (2005).</li><li>Mi, H., Muruganujan, A., Casagrande, J. T., Thomas, P. 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Dev. 76, 762-772 (2009).</li><li>Vallee, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Revealing+the+bovine+embryo+transcript+profiles+during+early+in+vivo+embryonic+development.">Revealing the bovine embryo transcript profiles during early in vivo embryonic development.</a> Reproduction. 138 (1), 95-105 (2009).</li><li>Dafforn, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Linear+mRNA+amplification+from+as+little+as+5+ng+total+RNA+for+global+gene+expression+analysis.">Linear mRNA amplification from as little as 5 ng total RNA for global gene expression analysis.</a> Biotechniques. 37 (5), 854-857 (2004).</li><li>Beaujean, N., Jammes, H., Jouneau, A., Dufort, I., Rovert, C., Sirard, M. 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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...

Early embryonic loss in high-producing dairy cows is one of the main challenges in the dairy industry1,2. Bovine has become an interesting model to study human preimplantation embryo development due to their similar developmental process3,4. 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 technology reduced printing errors and variability of array chips within and between different microarray platforms6. Improved microarray technology resulted in a widely application of this technology in clinical research7 and more recently, in early embryo quality assessment8.
The large amount of required material for microarray technology is the main reason why the microarray technology initially failed to enter a number of research fields such as early embryonic development. More recently, RNA amplification methods have been improved to linearly amplify RNA up to micrograms level from sub-nanogram starting RNA material9. There are several commercial RNA amplification kits available on the market; however, the more popular well developed kits are related to Ribo-Single Primer Isothermal Amplification10 and T7 promoter driven11 methods. The most popular antisense RNA amplification uses in vitro transcription with an oligo dT primer linking to a T7 promoter at 5&#39; end12. This technology allows maintaining the most of representative anti-sense transcripts after linear amplification for arrays hybridization13. This method has been adapted to amplify picogram level of total RNA extracted from bovine embryo8.
Universal Linkage System (ULS) is the labeling method which directly incorporates the DNA or amplified RNA with platinum-linked fluorescent dye either Cyanine 547 or Cyanine 647, by forming a coordinative bond on N7 position of guanine14. This method was adapted in embryos research to generate more stable amplified aRNA without modification compared to the aminoallyl modified aRNA generated by enzymatic method15. Both single dye and two dyes labeling methods have been adapted using Universal Linkage System in microarray. A large microarray comparisons study was that there is a good correlation of data quality between the one- and two-color array platforms6.
Recently, both T7 promoter driven antisense RNA amplification and ULS labeling methods have been developed to provide a more reliable protocol to generate a sufficient amount of high quality labeled aRNA materials for microarray hybridization8,16. Therefore, this study provides a protocol to demonstrate some of the important steps from RNA extraction to data analysis involved in two-color microarray using Day 7 bovine embryos as an example.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>PicoPure RNA Isolation Kit</td>
			<td>Applied Biosystems</td>
			<td>KIT0204</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase-Free DNase Set (50)</td>
			<td>Qiagen</td>
			<td>79254</td>
			<td></td>
		</tr>
		<tr>
			<td>Agilent RNA 6000 Pico Kit</td>
			<td>Agilent Technologies</td>
			<td>5067-1513</td>
			<td></td>
		</tr>
		<tr>
			<td>Arcturus RiboAmp HS PLUS Kit</td>
			<td>Applied Biosystems</td>
			<td>KIT0505</td>
			<td></td>
		</tr>
		<tr>
			<td>2100 Bioanalyzer Instruments</td>
			<td>Agilent Technologies</td>
			<td>G2940CA</td>
			<td></td>
		</tr>
		<tr>
			<td>RNA Screen Tape</td>
			<td>Agilent Technologies</td>
			<td>5067-5576</td>
			<td></td>
		</tr>
		<tr>
			<td>ULS Fluorescent Labeling Kit</td>
			<td>Kreatech Diagnostics</td>
			<td>EA-021</td>
			<td></td>
		</tr>
		<tr>
			<td>Custom Gene Expression Microarrays</td>
			<td>Agilent Technologies</td>
			<td>G2514F</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Agilent Gene Expression wash buffer 1</td>
			<td>Agilent Technologies</td>
			<td>Part #5188-5325</td>
			<td></td>
		</tr>
		<tr>
			<td>Agilent Gene Expression wash buffer 2</td>
			<td>Agilent Technologies</td>
			<td>Part #5188-5326</td>
			<td></td>
		</tr>
		<tr>
			<td>2X Hi-RPM Hybridization buffer</td>
			<td>Agilent Technologies</td>
			<td>&#160;Part #5190-0403</td>
			<td></td>
		</tr>
		<tr>
			<td>25X Fragment buffer</td>
			<td>Agilent Technologies</td>
			<td>Part #5185-5974</td>
			<td></td>
		</tr>
		<tr>
			<td>10X GE Blocking Agent</td>
			<td>Agilent Technologies</td>
			<td>Part #5188-5281</td>
			<td></td>
		</tr>
		<tr>
			<td>Stabilization and drying solution</td>
			<td>Agilent Technologies</td>
			<td>&#160;Part #5185-5979</td>
			<td></td>
		</tr>
		<tr>
			<td>Gasket slides enabled by Agilent SureHyb techonolgy</td>
			<td>Agilent Technologies</td>
			<td>G2524-60012</td>
			<td>Pack of 20 gasket slides, 4 microarrays/slide</td>
		</tr>
		<tr>
			<td>Two-Color RNA Spike-In Kit</td>
			<td>Agilent Technologies</td>
			<td>&#160;Cat# 5188-5279</td>
			<td></td>
		</tr>
		<tr>
			<td>GenePix 4000B array scanner</td>
			<td>Molecular Devices</td>
			<td>GENEPIX 4000B-U</td>
			<td></td>
		</tr>
		<tr>
			<td>Ozone Free Box</td>
			<td>BioTray</td>
			<td>OFB_100x200</td>
			<td></td>
		</tr>
		<tr>
			<td>GAL file</td>
			<td>Agilent Technologies</td>
			<td>-</td>
			<td></td>
		</tr>
	</tbody>
</table>

transcriptome profiling of in-vivo produced bovine pre-implantation embryos using two-color microarray platform

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.

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...

Watch this Scientific Journal Video about Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform at JoVE.com

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

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.

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

Early embryonic loss is a large contributor to infertility in cattle. Moreover, bovine becomes an interesting model to study human preimplantation embryo ...

The overall goal of this video is to provide an optimized technical procedure using a two-color custom-made bovine array using a low amount of total RNA from day seven bovine embryos. This method can help answer key questions regarding embryonic loss in high-producing dairy cows and questions about embryonic quality relative to gene expression in the developing embryo prior to implantation. The advantage of this technique is that we can study gene expression using picogram level of total RNA extracted from single embryos.This method can provide insight into the factor affecting the survival of in vivo produced bovine pre-implantation embryos. This can also help improve reproductive technologies such as in vitro embryo productions. To begin the procedure, prepare snap frozen bovine embryos in a microcentrifuge tube from a column-based pico scale RNA extraction kit.Add 10 microliters of lysis buffer to the tube, and incubate the embryos at 42 degrees Celsius for 30 minutes. Then, centrifuge the tube for two minutes at 800 times G.Pipette 250 microliters of conditioning buffer into the purification column filter membrane, and incubate the column for five minutes at room temperature. Centrifuge the collection tube at 16, 000 times G for one minute to finish preconditioning the column.Add 10 microliters of 70%ethanol to the mixture of lysis buffer and embryos and mix well. Then, load the mixture onto the purification column. Centrifuge the column for two minutes at 100 times G, and then at 16, 000 times G for 30 seconds.Add 100 microliters of the first wash buffer to the column, and centrifuge at 8, 000 times G for one minute. Using a DNase kit, mix five microliters of stock solution with 35 microliters of buffer, and mix by gently inverting the closed tube. Then, load the DNase mixture onto the purification column membrane, and incubate for 15 minutes at room temperature.Add 40 microliters of the first wash buffer to the column, and centrifuge at 8, 000 times G for 15 seconds. Then, add 100 microliters of the second wash buffer, and centrifuge for a minute. Add another portion of the second wash buffer to the column, and centrifuge for two minutes at 16, 000 times G.Transfer the purification column to another microcentrifuge tube, and load 11 microliters of elution buffer onto the column membrane.Incubate the column for one minute at room temperature. Centrifuge the column for one minute at 1, 000 times G, and then for another minute at 16, 000 times G.Check the quality and quantity of the eluted RNA, and then store the sample in negative 80 degrees Celsius. Using an amplification kit, amplify 100 picograms of high-quality RNA.Evaluate the size range of the amplified aRNA with fluorescence-based quantitation. Then, determine the concentration of aRNA by spectrophotometry. Prepare two micrograms of the amplified aRNA for fluorescent labeling with a standard kit.Set up an ozone box in a darkroom, and wait until the ozone level decreases to 0.001 parts per million. Place a darkroom filter over the light. Then, bring the sample into the ozone box and add two microliters each of cyanine five dye and labeling buffer.With the sample under a safe light, add RNase free water to achieve a total volume of 20 microliters. Gently mix the sample, and then, incubate the tube in a thermocycler at 85 degrees Celsius for 15 minutes. Cool the sample on ice for one minute, and then, spin down the sample.Clean up the labeled and amplified aRNA with an RNA extraction kit. Using the appropriate type of spectrometer, determine the concentration of labeled, amplified aRNA. Prepare a second sample of amplified aRNA labeled with cyanine three dye.Store the aRNA samples at negative 80 degrees Celsius for up to three days. Combine 825 nanograms each of Cy3 and 5-labeled aRNA. Add to this the blocking buffer and the fragmentation buffer.Incubate the mixture at 60 degrees Celsius for 15 minutes, and then, cool on ice for one minute. Add 55 microliters hybridization buffer, and mix well without introducing air bubbles. Centrifuge the mixture at 11, 300 times G for one minute.Load 100 microliters of sample onto the array slide, and seal the slide in the hybridization chamber. Hybridize the sample for 17 hours at 65 degrees Celsius while rotating at 10 rotations per minute. Prepare an ozone-scavenging stabilizing and drying solution.Wash the arrays, taking precautions to minimize exposure to ozone, and then, immerse them in the stabilizing and drying solution for 30 seconds. Ensure the array surface is dry and free of dust. Store the arrays in a dark location.Turn on the microarray scanner, and wait until it is ready to scan. Then, load the array with the barcode to the left. Perform a spot analysis, and save the data as a GPR file.In statistical analysis software, normalize and analyze the data. Calculate the positive spot selection threshold, and view the plot of hybridized and background signals. Perform a Loess normalization within array, and then a quantile between arrays normalization.Export the normalized data to a spreadsheet file. Use the all positive signals in a microarray data repository to create an ID list of selected unique genes. Upload the ID list to a protein classification and analysis database, choosing bos taurus as the organism, and perform a default statistical overrepresentation test.Following two-round amplification, the fragments are very similar in size and of good quality. Successful amplification by this method should yield at least a thousand-fold increase of aRNA. A high-quality microarray image has high spot intensity and low background intensity on both channels.If the background intensity is too high, signal resolution will be poor. About 46%of the spots were above background, from which 6, 765 unique RNA transcripts expressed in the blastocyst were isolated. A statistical overrepresentation test indicated that the top 10 gene ontology terms represented active cellular division processes.The development of this technique has allowed researchers in the area of animal reproduction to explore embryonic gene expression and evaluate how various factors effect the developing embryo. This technique has also been developed in other important species, such as the pig. Following this procedure, other methods such as quantitative PCR can be used to validate microarray results.After watching this video, you should have a good understanding of how to extract RNA from embryos as well as how to amplify and label RNA to perform two-color microarray analysis.

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Research

JoVE Journal

Developmental Biology

7.7K visualizações.  University of Alberta. The overall goal of this video is to provide an optimized technical procedure using a two-color custom-made bovine array using a low amount of total RNA from day seven bovine embryos. This method can help answer key questions regarding embryonic loss in high-producing dairy cows and questions about embryonic quality relative to gene expression in the developing embryo prior to implantation. The advantage of this technique is that we can study gene expression using picogram level of total RNA ...