Name: maintaining biological cultures and measuring gene expression in aphis nerii: a non-model system for plant-insect interactions
Uploaded: 2018-08-31T14:00:00
Description: Watch this Scientific Journal Video about Maintaining Biological Cultures and Measuring Gene Expression in Aphis nerii: A Non-model System for Plant-insect Interactions at JoVE.com

We would like to thank Michelle Moon (Vanderbilt University) for assistance with photography. Vanderbilt University provided support to PA and SSLB is supported by DGE-1445197.

1. Plant Cultures
<ol>
	<li>Purchase seeds from any commercial vendor or collect from mature plants in the field. 
	NOTE: This protocol is suitable for most commercially available milkweed species (e.g., Asclepias incarnata, A. syriaca, A. curassavica, Gomphocarpus physocarpus). Some seeds may need to be cold-stratified, and instructions from the seed supplier should be checked.</li>
	<li>Plant seeds in a fine germinating soil (60&#8211;70% fine peat moss, perlite, vermi...

<ol>
	<li>Brisson, J. A., Stern, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pea+aphid,+Acyrthosiphon+pisum.:+an+emerging+genomic+model+system+for+ecological,+developmental+and+evolutionary+studies.">The pea aphid, Acyrthosiphon pisum.: an emerging genomic model system for ecological, developmental and evolutionary studies.</a> BioEssays. 28, 747-755 (2006).</li><li>Dixon, A. F. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Aphid Ecology: An Optimization Approach. , (1985).</li><li>Consortium, T. I. A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+Sequence+of+the+Pea+Aphid+Acyrthosiphon+pisum.">Genome Sequence of the Pea Aphid Acyrthosiphon pisum.</a> PLoS Biology. , 1000313 (2010).</li><li>Srinivasan, D. G., Brisson, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aphids:+A+Model+for+Polyphenism+and+Epigenetics.">Aphids: A Model for Polyphenism and Epigenetics.</a> Genetics Research International. 2012, 1-12 (2012).</li><li>Wenger, J. 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=Whole+genome+sequence+of+the+soybean+aphid,+Aphis+glycines.">Whole genome sequence of the soybean aphid, Aphis glycines.</a> Insect Biochememistry and Molecular Biology. , 1-10 (2017).</li><li>Li, Z. -. Q., 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=Ecological+adaption+analysis+of+the+cotton+aphid+(Aphis+gossypii)+in+different+phenotypes+by+transcriptome+comparison.">Ecological adaption analysis of the cotton aphid (Aphis gossypii) in different phenotypes by transcriptome comparison.</a> PLoS ONE. 8, 83180 (2013).</li><li>Wang, D., Liu, Q., Jones, H. D., Bruce, T., Xia, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+transcriptomic+analyses+revealed+divergences+of+two+agriculturally+important+aphid+species.">Comparative transcriptomic analyses revealed divergences of two agriculturally important aphid species.</a> BMC Genomics. 15 (1), 1023-1024 (2014).</li><li>Wu, S., 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=De+novo+characterization+of+the+pine+aphid+Cinara+pinitabulaeformis+Zhang+et+Zhang+transcriptome+and+analysis+of+genes+relevant+to+pesticides.">De novo characterization of the pine aphid Cinara pinitabulaeformis Zhang et Zhang transcriptome and analysis of genes relevant to pesticides.</a> PLoS ONE. 12, 0178496-0178517 (2017).</li><li>Birnbaum, S. S. L., Rinker, D. C., Gerardo, N. 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G., Rasmann, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxic+cardenolides:+chemical+ecology+and+coevolution+of+specialized+plant-herbivore+interactions.">Toxic cardenolides: chemical ecology and coevolution of specialized plant-herbivore interactions.</a> New Phytologist. 194, 28-45 (2012).</li><li>Dobler, S., Petschenka, G., Pankoke, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coping+with+toxic+plant+compounds-+the+insect's+perspective+on+iridoid+glycosides+and+cardenolides.">Coping with toxic plant compounds- the insect's perspective on iridoid glycosides and cardenolides.</a> Phytochemistry. 72, 1593-1604 (2011).</li><li>Opitz, S. E. W., M&#252;ller, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+chemistry+and+insect+sequestration.">Plant chemistry and insect sequestration.</a> Chemoecology. 19, 117-154 (2009).</li><li>Birnbaum, S. S. L., Abbot, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insect+adaptations+toward+plant+toxins+in+milkweed-herbivores+systems+-+a+review.">Insect adaptations toward plant toxins in milkweed-herbivores systems - a review.</a> Entomologia Experimentalis et Applicata. 58, 579-610 (2018).</li><li>Rothschild, M., von Euw, J., Reichstein, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+glycosides+in+the+oleander+aphid,+Aphis+nerii.">Cardiac glycosides in the oleander aphid, Aphis nerii.</a> Journal of Insect Physiology. 16, 1141-1145 (1970).</li><li>Harrison, J. S., Mondor, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+an+invasive+aphid+'superclone':+extremely+low+genetic+diversity+in+oleander+aphid+(Aphis+nerii)+populations+in+the+southern+United+States.">Evidence for an invasive aphid 'superclone': extremely low genetic diversity in oleander aphid (Aphis nerii) populations in the southern United States.</a> PLoS ONE. 6, 17524 (2011).</li><li>Mooney, K. A., Halitschke, R., Kessler, A., Agrawal, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolutionary+trade-offs+in+plants+mediate+the+strength+of+trophic+cascades.">Evolutionary trade-offs in plants mediate the strength of trophic cascades.</a> Science. 327, 1642-1644 (2010).</li><li>Grabherr, M. G., 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=Full-length+transcriptome+assembly+from+RNA-Seq+data+without+a+reference+genome.">Full-length transcriptome assembly from RNA-Seq data without a reference genome.</a> Nature Biotechnology. 29, 644-652 (2011).</li><li>Haas, B. J., 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=De+novo+transcript+sequence+reconstruction+from+RNA-seq+using+the+Trinity+platform+for+reference+generation+and+analysis.">De novo transcript sequence reconstruction from RNA-seq using the Trinity platform for reference generation and analysis.</a> Nature Protocols. 8, 1494-1512 (2013).</li><li>Finn, R. 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=The+Pfam+protein+families+database:+towards+a+more+sustainable+future.">The Pfam protein families database: towards a more sustainable future.</a> Nucleic Acids Research. 44, 279-285 (2016).</li><li>The UniProt Consortium. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=UniProt:+the+universal+protein+knowledgebase.">UniProt: the universal protein knowledgebase.</a> Nucleic Acids Research. 45, 158-169 (2017).</li><li>Johnson, 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=NCBI+BLAST:+a+better+web+interface.">NCBI BLAST: a better web interface.</a> Nucleic Acids Research. 1 (36), 5-9 (2008).</li><li>Fu, L., Niu, B., Zhu, Z., Wu, S., Li, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD-HIT:+accelerated+for+clustering+the+next+generation+sequencing+data.">CD-HIT: accelerated for clustering the next generation sequencing data.</a> Bioinformatics. 28 (23), 3150-3152 (2012).</li><li>Sim&#227;o, F. A., Waterhouse, R. M., Ioannidis, P., Kriventseva, E. V., Zdobnov, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BUSCO:+assessing+genome+assembly+and+annotation+completeness+with+single-copy+orthologs.">BUSCO: assessing genome assembly and annotation completeness with single-copy orthologs.</a> Bioinformatics. 31, 3210-3212 (2015).</li><li>Bolger, A. M., Lohse, M., Usadel, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trimmomatic:+a+flexible+trimmer+for+Illumina+sequence+data.">Trimmomatic: a flexible trimmer for Illumina sequence data.</a> Bioinformatics. 30, 2114-2120 (2014).</li><li>Langmead, B., Salzberg, S. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+quantitative+RT-PCR:+design,+calculations,+and+statistics.">Real-time quantitative RT-PCR: design, calculations, and statistics.</a> Plant Cell. 21, 1031-1033 (2009).</li><li>Malcolm, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+defence+in+chewing+and+sucking+insect+herbivores:+plant-derived+cardenolides+in+the+monarch+butterfly+and+oleander+aphid.">Chemical defence in chewing and sucking insect herbivores: plant-derived cardenolides in the monarch butterfly and oleander aphid.</a> Chemoecology. 1, 12-21 (1990).</li><li>Agrawal, A. A., Underwood, N., Stinchcombe, J. 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It has long been recognized that the aposematic A. nerii can provide insights into the patterns and mechanisms of resistance to plant defenses and particularly chemical sequestration18,37. A number of genomic resources have recently emerged for A. nerii10, offering new opportunities for ecological and functional genomic studies that use A. nerii as a model. We outline basic protocols in aphid and plant culture, a...

Aphids are small, hemimetabolous insects that colonize on diverse plant families worldwide. They are distinctive for several features, most notably their complex life cycles involving cyclical parthenogenesis and discrete polyphenisms, and their obligate nutritional symbioses with bacterial or yeast endosymbionts that supply nutrients missing from their diet of plant sap1. While most aphids are host plant specialists, some generalist species are important crop pests, inflicting considerable economic damage on crops either directly or via the pathogens and viruses they vector2. The publication of the first aphid genome in 2010, the pea aphid Acyrthosiphon pisum3, marked an important milestone in the study of aphid biology because it provided the genomic resources for addressing questions about the insect&#39;s adaptations to the herbivorous lifestyles, including those that might lead to a better control strategies4. Since that time, additional genomic resources have accumulated with the publication of an annotated genome for the soybean aphid Aphis glycines5, and publicly-available whole genome resources for another three-aphid species (Myzus cerasi (black cherry aphid), Myzus persicae (peach-potato aphid), Rhopalosiphum padi (bird cherry-oat aphid)6. Valuable de novo transcriptomic resources are available as well for a number of other aphid species (e.g.,Aphis gossypii (cotton aphid)7, Sitobion avenae (grain aphid)8, Cinara pinitabulaeformis (pine aphid)9, Aphis nerii (milkweed-oleander aphid)10).
Aphids have also made lasting contributions to our understanding of the plant-insect interactions and the ecology of the life on plants11. One area where aphids have made particularly important contributions is in our understanding of the chemical ecology of the host plant interactions. Herbivorous insects express diverse adaptations for overcoming plant defenses, and some even co-opt plant defenses for their own benefit12,13,14. For example, the milkweed-oleander aphid, Aphis nerii, is a bright yellow, invasive aphid found in temperate and tropical regions worldwide that colonizes on plants in the milkweed family (Apocynaceae). Plants in the family Apocynaceae have evolved diverse chemical defenses, including milky latex and cardiac glycosides known as cardenolides, that bind the cation carrier Na,K-ATPase and are effective deterrents to generalist herbivores15,16. Milkweed specialists express various modes of resistance to cardenolides, and some selectively or passively accumulate or modify cardenolides in their tissues as a means to deter predation or for other benefits17. A. nerii is thought to sequester cardenolides in this way, although the mechanisms and functional benefits remain unclear10,18.
Given the genomic resources at hand, A. nerii provides an excellent experimental model for the study of the molecular and genetic mechanisms involved in the chemo-ecological interactions between toxic host plants and their specialist herbivores. It is worth noting that, while some of the earliest studies of A. nerii focused on sequestration of cardenolides19, since that time, studies of A. nerii have provided insights into a broad set of evolutionary and ecological questions, including the genetic structure of invasive insects20 and the interplay between bottom-up and top-down regulation on the herbivore density21. A. nerii is thus a good candidate as an experimental model for an especially broad set of studies of the insect-plant interactions. Critical to the success of any study with A. nerii is the careful culture of aphid populations, which includes the culture of plants on which the aphids depend, as well as an efficient generation of high-quality -omic data. Our goal is to guide the reader through both. Outlined below are methods for the generation and maintenance of the plant and aphid cultures in the greenhouse and laboratory, DNA and RNA extractions, microsatellite analysis, de novo transcriptome assembly and annotation, transcriptome differential expression analysis, and qPCR verification of differentially expressed genes. While these methods are written for A. nerii, the general culturing, extraction, and analysis methods can extend to a variety of aphid species.

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	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="84">
			<td height="84" style="height:84px;width:64px;">Sun Gro Fafard Germination Mix</td>
			<td style="width:160px;">Hummert International</td>
			<td style="width:184px;">10-0952-2</td>
			<td style="width:412px;"></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:64px;">Sun Gro Fafard 3B/ Metro Mix</td>
			<td>Hummert International</td>
			<td style="width:184px;">10-0951-2</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:64px;">2x 4&#34; Round Standard Pot</td>
			<td>Anderson Pots</td>
			<td style="width:184px;">1503</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:64px;">DreamTaq DNA Polymerase</td>
			<td>ThermoFisher Scientific</td>
			<td style="width:184px;">EP0701</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:64px;">Trizol</td>
			<td>ThermoFisher Scientific</td>
			<td style="width:184px;">15596026</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SuperScript&#174; III First-Strand Synthesis kit</td>
			<td>ThermoFisher Scientific</td>
			<td style="width:184px;">18080051</td>
			<td></td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:64px;">Power SYBR Green PCR Master Mix</td>
			<td>ThermoFisher Scientific</td>
			<td style="width:184px;">4367659</td>
			<td></td>
		</tr>
	</tbody>
</table>

maintaining biological cultures and measuring gene expression in aphis nerii: a non-model system for plant-insect interactions

Aphids are excellent experimental models for a variety of biological questions ranging from the evolution of symbioses and the development of polyphenisms to questions surrounding insect's interactions with their host plants. Genomic resources are available for several aphid species, and with advances in the next-generation sequencing, transcriptomic studies are being extended to non-model organisms that lack genomes. Furthermore, aphid cultures can be collected from the field and reared in the laboratory for the use in organismal and molecular experiments to bridge the gap between ecological and genetic studies. Last, many aphids can be maintained in the laboratory on their preferred host plants in perpetual, parthenogenic life cycles allowing for comparisons of asexually reproducing genotypes. Aphis nerii, the milkweed-oleander aphid, provides one such model to study insect interactions with toxic plants using both organismal and molecular experiments. Methods for the generation and maintenance of the plant and aphid cultures in the greenhouse and laboratory, DNA and RNA extractions, microsatellite analysis, de novo transcriptome assembly and annotation, transcriptome differential expression analysis, and qPCR verification of differentially expressed genes are outlined and discussed here.

Plant cultures: Seeds will take approximately two to four weeks, depending on the season, to grow large enough to be re-potted (Figure 1A). Re-potted seedlings will take another two to four weeks to grow to an optimal size for aphid cultures (Figure 1B).
Aphid cultures: Adult A. nerii are disti...

Watch this Scientific Journal Video about Maintaining Biological Cultures and Measuring Gene Expression in Aphis nerii: A Non-model System for Plant-insect Interactions at JoVE.com

Maintaining Biological Cultures and Measuring Gene Expression in Aphis nerii: A Non-model System for Plant-insect Interactions

The aphid Aphis nerii colonizes on highly-defended plants in the dogbane family (Apocyanaceae) and provides numerous opportunities to study plant-insect interactions. Here, we present a series of protocols for the maintenance of plant and aphid cultures, and the generation and analysis of molecular and -omic data for A. nerii.

Maintaining Biological Cultures and Measuring Gene Expression in Aphis nerii: A Non-model System for Plant-insect Interactions

Aphids are excellent experimental models for a variety of biological questions ranging from the evolution of symbioses and the development of polyphenisms ...

This method describes general steps for aphid culturing and steps for the genetic analysis of aphids. These methods can be applied to answer key questions in the evolution and ecology of aphids and the moleculare mechanisms underlying aphid biology. The main advantage of this technique is it can be generally applied to most aphid species and performed at a relatively low cost.Begin by filling a standard seedling tray with germination mix soil, taking care that the soil reaches the top of the wells. When all of the wells have been filled, make a three centimeter deep hole in the soil of each well and place one seed into each hole. Water the cells until the soil covering the seeds is saturated and place the tray in a green house with daily to every other day watering, to maintain a moderate soil moisture level.When the seedlings have grown their first set of full leaves fill four inch round pots with potting soil, up to about five centimeters below their rims. Create a whole in the soil deep enough to reach the bottom of the pot and gently scoop the mature seedlings by hand to place them deep within the holes in the new pots. Then, cover the seedlings with soil and water the seedlings daily to every other day to maintain a moderate soil moisture level.When the plants have grown at least three to four sets of full leaves and are at least ten centimeters tall, manually inspect the plants for any unwanted pests and use a mouth pipette to carefully transfer a single reproducing field collected adult aphid onto the leaf of the first plant to create an iso-clonal line. When all of the aphids have been transferred, securely cover the plants with a custom made cup cage, and place the aphid infested plants in a controlled environment chamber. To maintain the stock populations, use a mouth pipette to safely transfer one to three second or third instar nymphs, and one adult aged aphid, to a new plant on a weekly basis.And cover each plant with a new cup cage for culture as demonstrated. For DNA extraction from a single adult aphid, place the aphid near the bottom of a sterile 1.5 milliliter microcentrifuge tube containing liquid nitrogen and grind the aphid with a pestle. Next, add 100 microliters of lysis buffer to the microcentrifuge tube and continue to grind the crushed aphid until the sample is visibly disintegrated.Wash the pestle with an additional 100 microliters of lysis buffer, and incubate the ground aphid at 65 degrees Celsius for 30 minutes. At the end of the incubation add 14 microliters of aemulor potassium acetate to the tube and invert to mix. After 30 minutes on ice collect the cellular debris by centrifugation and transfer the supernatant to a new 1.5 milliliter microcentrifuge tube without disturbing the pellet.Add 200 microliters of cold 100 percent molecular grade ethanol to the supernatant and invert the tube to mix. After 15 minutes at room temperature, collect the precipitated DNA by centrifugation, and remove the ethanol by pipette. Resuspend the pellet in 200 microliters of cold 70 percent molecular grade ethanol with flicking for a second centrifuge wash.And resuspend the pellet in 200 microliters of fresh, cold 100 percent molecular grade ethanol. After the third centrifugation, place the tube horizontally on a piece of tissue paper for air drying for five to ten minutes. And resuspend the DNA pellet in 80 microliters of low tris edta.Then, quantify the resuspended DNA in a spectrophotometer, and store the remaining sample at four degrees Celsius. After crushing up to five adult aphids in liquid nitrogen as demonstrated, transfer the sample to a fume hood and add 800 microliters of guanidinium thiocyanate-phenol-chloroform extraction reagent to the sample tube. Homogenize the sample with a pestle and incubate the homogenate for five minutes at room temperature.Next, add 160 microliters of chloroform to the sample, followed by 15 seconds of vigorous shaking by hand. Then, incubate the sample for two to three minutes at room temperature and centrifuge to collect the extracted RNA. To precipitate the RNA, transfer the aqueous phase to new RNS free microcentrifuge tube without disturbing the intermediate phase, and add 400 microliters of isopropanol to the RNA.After a ten minute incubation at negative 20 degrees Celsius, collect the precipitated RNA by centrifugation. Then, wash the sample two times with one milliliter of 75 percent ethanol in diethyl pyrocarbonate treated water, to the sample with slow vortexing and centrifugation to remove any phenol contaminants. After the second wash, mostly air dry the pellet for five to ten minutes on a sterile bench, and dissolve the RNA pellet in 30 microliters of ultra pure water with gentle pipetting before a ten to 15 minute incubation at 55 to 60 degrees Celsius.Seeds will take approximately two to four weeks, depending on the season, to grow large enough to be repotted. Repotted seedlings will take another two to four weeks to grow to an optimal size for aphid culture. Adult aye-near-ee-eye are distinguished by a darkened cauda, and may be unwinged or winged.Developing wing pads become visible when nymphs reach the third instar. Single adult aye-near-ee-eye will yield approximately 100 to 200 nanograms per microliter of DNA and 150 to 300 nanograms per microliter of RNA. The relative expression of a candidate gene under different treatment conditions can then be calculated.When attempting this procedure it's important to not contaminate you're iso-clonal stocks. Also, remember that the quality of your aphids is highly dependent upon the quality of your plants. Following this procedure, other extractions and sequencing techniques can be performed to answer question about the molecular mechanisms underlying aphid biology.

maintaining-biological-cultures-measuring-gene-expression-aphis-nerii

Research

JoVE Journal

Genetics

Parallel Measurement of Circadian Clock Gene Expression and Hormone Secretion in Human Primary Cell Cultures

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental changes. Considerable progress in our understanding of the tight connection between the circadian clock and most aspects of physiology has been made in the field over the last decade. However, unraveling the molecular basis that underlies the function of the circadian oscillator in humans stays of highest technical challenge. Here, we provide a detailed description of an experimental approach for long-term (2-5 days) bioluminescence recording and outflow medium collection in cultured human primary cells. For this purpose, we have transduced primary cells with a lentiviral luciferase reporter that is under control of a core clock gene promoter, which allows for the parallel assessment of hormone secretion and circadian bioluminescence. Furthermore, we describe the conditions for disrupting the circadian clock in primary human cells by transfecting siRNA targeting CLOCK. Our results on the circadian regulation of insulin secretion by human pancreatic islets, and myokine secretion by human skeletal muscle cells, are presented here to illustrate the application of this methodology. These settings can be used to study the molecular makeup of human peripheral clocks and to analyze their functional impact on primary cells under physiological or pathophysiological conditions.

Here, we describe settings to monitor in parallel circadian bioluminescence and the secretory activity of human islet cells and primary myotubes. For this, we employed lentiviral gene delivery of a luciferase core clock reporter, followed by in vitro synchronization and collection of outflow medium by continuous cell perifusion.

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental ...

Perturbations of Circulating miRNAs in Irritable Bowel Syndrome Detected Using a Multiplexed High-throughput Gene Expression Platform

The gene expression platform assay allows for robust and highly reproducible quantification of the expression of up to 800 transcripts (mRNA or miRNAs) in a single reaction. The miRNA assay counts transcripts by directly imaging and digitally counting miRNA molecules that are labeled with color-coded fluorescent barcoded probe sets (a reporter probe and a capture probe). Barcodes are hybridized directly to mature miRNAs that have been elongated by ligating a unique oligonucleotide tag (miRtag) to the 3&#39; end. Reverse transcription and amplification of the transcripts are not required. Reporter probes contain a sequence of six color positions populated using a combination of four fluorescent colors. The four colors over six positions are used to construct a gene-specific color barcode sequence. Post-hybridization processing is automated on a robotic prep station. After hybridization, the excess probes are washed away, and the tripartite structures (capture probe-miRNA-reporter probe) are fixed to a streptavidin-coated slide via the biotin-labeled capture probe. Imaging and barcode counting is done using a digital analyzer. The immobilized barcoded miRNAs are visualized and imaged using a microscope and camera, and the unique barcodes are decoded and counted. Data quality control (QC), normalization, and analysis are facilitated by a custom-designed data processing and analysis software that accompanies the assay software. The assay demonstrates high linearity over a broad range of expression, as well as high sensitivity. Sample and assay preparation does not involve enzymatic reactions, reverse transcription, or amplification; has few steps; and is largely automated, reducing investigator effects and resulting in high consistency and technical reproducibility. Here, we describe the application of this technology to identifying circulating miRNA perturbations in irritable bowel syndrome.

We describe the use of a multiplexed high-throughput gene expression platform that quantitates gene expression by barcoding and counting molecules in biological substrates without the need for amplification. We used the platform to quantitate microRNA (miRNA) expression in whole blood in subjects with and without irritable bowel syndrome.

The gene expression platform assay allows for robust and highly reproducible quantification of the expression of up to 800 transcripts (mRNA or miRNAs) in ...

Single-cell Gene Expression Profiling Using FACS and qPCR with Internal Standards

Gene expression measurements from bulk populations of cells can obscure the considerable transcriptomic variation of individual cells within those populations. Single-cell gene expression measurements can help assess the role of noise in gene expression, identify correlations in the expression of pairs of genes, and reveal subpopulations of cells that respond differently to a stimulus. Here, we describe a procedure to measure the expression of up to 96 genes in single mammalian cells isolated from a population growing in tissue culture. Cells are sorted into lysis buffer by fluorescence-activated cell sorting (FACS), and the mRNA species of interest are reverse-transcribed and amplified. Gene expression is then measured using a microfluidic real-time PCR machine, which performs up to 96 qPCR assays on up to 96 samples at a time. We also describe the generation and use of PCR amplicon standards to enable the estimation of the absolute number of each transcript. Compared with other methods of measuring gene expression in single cells, this approach allows for the quantification of more distinct transcripts than RNA FISH at a lower cost than RNA-Seq.

We describe a method to sort single mammalian cells and to quantify the expression of up to 96 target genes of interest in each cell. This method includes the use of internal qPCR standards to enable the estimation of absolute transcript counts.

Gene expression measurements from bulk populations of cells can obscure the considerable transcriptomic variation of individual cells within those ...

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

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.

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.

The analysis of global gene expression changes is a valuable tool for identifying novel pathways underlying observed phenotypes. The zebrafish is an ...

Measuring Gene Expression in Bombarded Barley Aleurone Layers with Increased Throughput

The aleurone layer of barley grains is an important model system for hormone-regulated gene expression in plants. In aleurone cells, genes required for germination or early seedling development are activated by gibberellin (GA), while genes associated with stress responses are activated by abscisic acid (ABA). The mechanisms of GA and ABA signaling can be interrogated by introducing reporter gene constructs into aleurone cells via particle bombardment, with the resulting transient expression measured using enzyme assays. An improved protocol is reported that partially automates and streamlines the grain homogenization step and the enzyme assays, allowing significantly more throughput than existing methods. Homogenization of the grain samples is carried out using an automated tissue homogenizer, and GUS (&#946;-glucuronidase) assays are carried out using a 96-well plate system. Representative results using the protocol suggest that phospholipase D activity may play an important role in the activation of HVA1 gene expression by ABA, through the transcription factor TaABF1.

An improved protocol is presented for the measurement of transient gene expression from reporter constructs in barley aleurone cells after particle bombardment. The combination of automated grain grinding with 96-well plate enzyme assays provides high throughput for the procedure.

The aleurone layer of barley grains is an important model system for hormone-regulated gene expression in plants. In aleurone cells, genes required for ...

Efficient Production and Identification of CRISPR/Cas9-generated Gene Knockouts in the Model System Danio rerio

Characterization of the clustered, regularly interspaced, short, palindromic repeat (CRISPR) system of Streptococcus pyogenes has enabled the development of a customizable platform to rapidly generate gene modifications in a wide variety of organisms, including zebrafish. CRISPR-based genome editing uses a single guide RNA (sgRNA) to target a CRISPR-associated (Cas) endonuclease to a genomic DNA (gDNA) target of interest, where the Cas endonuclease generates a double-strand break (DSB). Repair of DSBs by error-prone mechanisms lead to insertions and/or deletions (indels). This can cause frameshift mutations that often introduce a premature stop codon within the coding sequence, thus creating a protein-null allele. CRISPR-based genome engineering requires only a few molecular components and is easily introduced into zebrafish embryos by microinjection. This protocol describes the methods used to generate CRISPR reagents for zebrafish microinjection and to identify fish exhibiting germline transmission of CRISPR-modified genes. These methods include in vitro transcription of sgRNAs, microinjection of CRISPR reagents, identification of indels induced at the target site using a PCR-based method called a heteroduplex mobility assay (HMA), and characterization of the indels using both a low throughput and a powerful next-generation sequencing (NGS)-based approach that can analyze multiple PCR products collected from heterozygous fish. This protocol is streamlined to minimize both the number of fish required and the types of equipment needed to perform the analyses. Furthermore, this protocol is designed to be amenable for use by laboratory personal of all levels of experience including undergraduates, enabling this powerful tool to be economically employed by any research group interested in performing CRISPR-based genomic modification in zebrafish.

Efficient Production and Identification of CRISPR/Cas9-generated Gene Knockouts in the Model System Danio rerio

Targeted genome editing in the model system Danio rerio (zebrafish) has been greatly facilitated by the emergence of CRISPR-based approaches. Herein, we describe a streamlined, robust protocol for generation and identification of CRISPR-derived nonsense alleles that incorporates the heteroduplex mobility assay and identification of mutations using next-generation sequencing.

Characterization of the clustered, regularly interspaced, short, palindromic repeat (CRISPR) system of Streptococcus pyogenes has enabled the development ...

An Optogenetic Method to Control and Analyze Gene Expression Patterns in Cell-to-cell Interactions

Cells should respond properly to temporally changing environments, which are influenced by various factors from surrounding cells. The Notch signaling pathway is one of such essential molecular machinery for cell-to-cell communications, which plays key roles in normal development of embryos. This pathway involves a cell-to-cell transfer of oscillatory information with ultradian rhythms, but despite the progress in molecular biology techniques, it has been challenging to elucidate the impact of multicellular interactions on oscillatory gene dynamics. Here, we present a protocol that permits optogenetic control and live monitoring of gene expression patterns in a precise temporal manner. This method successfully revealed that intracellular and intercellular periodic inputs of Notch signaling entrain intrinsic oscillations by frequency tuning and phase shifting at the single-cell resolution. This approach is applicable to the analysis of the dynamic features of various signaling pathways, providing a unique platform to test a functional significance of dynamic gene expression programs in multicellular systems.

Here, we present a protocol to analyze cell-to-cell transfer of oscillatory information by optogenetic control and live monitoring of gene expression. This approach provides a unique platform to test a functional significance of dynamic gene expression programs in multicellular systems.

Cells should respond properly to temporally changing environments, which are influenced by various factors from surrounding cells. The Notch signaling ...

An Ecdysone Receptor-based Singular Gene Switch for Deliberate Expression of Transgene with Robustness, Reversibility, and Negligible Leakiness

Precise control of transgene expression is desirable in biological and clinical studies. However, because the binary feature of currently employed gene switches requires the transfer of two therapeutic expression units concurrently into a single cell, the practical application of the system for gene therapy is limited. To simplify the transgene expression system, we generated a gene switch designated as pEUI(+) encompassing a complete set of transgene expression modules in a single vector. Comprising of the GAL4 DNA-binding domain and modified EcR (GvEcR), a minimal VP16 activation domain fused with a GAL4 DNA-binding domain, as well as a modified Drosophila ecdysone receptor (EcR), the newly developed singular gene switch is highly responsive to the administration of a chemical inducer in a time- and dosage-dependent manner. The pEUI(+) vector is a potentially powerful tool for improving the control of transgene expression in both biological research and pre-clinical studies. Here, we present a detailed protocol for modulation of a transient and stable transgene expression using pEUI(+) vector by the treatment of tebufenozide (Teb). Additionally, we share important guidelines for the use of Teb as a chemical inducer.

Here, we present a protocol for the modulation of transgene expression using pEUI(+) singular gene switch by tebufenozide treatment.

Precise control of transgene expression is desirable in biological and clinical studies. However, because the binary feature of currently employed gene ...

Exploring the Root Microbiome: Extracting Bacterial Community Data from the Soil, Rhizosphere, and Root Endosphere

The intimate interaction between plant host and associated microorganisms is crucial in determining plant fitness, and can foster improved tolerance to abiotic stresses and diseases. As the plant microbiome can be highly complex, low-cost, high-throughput methods such as amplicon-based sequencing of the 16S rRNA gene are often preferred for characterizing its microbial composition and diversity. However, the selection of appropriate methodology when conducting such experiments is critical for reducing biases that can make analysis and comparisons between samples and studies difficult. This protocol describes in detail a standardized methodology for the collection and extraction of DNA from soil, rhizosphere, and root samples. Additionally, we highlight a well-established 16S rRNA amplicon sequencing pipeline that allows for the exploration of the composition of bacterial communities in these samples, and can easily be adapted for other marker genes. This pipeline has been validated for a variety of plant species, including sorghum, maize, wheat, strawberry, and agave, and can help overcome issues associated with the contamination from plant organelles.

Here, we describe a protocol to obtain amplicon sequence data of soil, rhizosphere, and root endosphere microbiomes. This information can be used to investigate the composition and diversity of plant-associated microbial communities, and is suitable for the use with a wide range of plant species.

The intimate interaction between plant host and associated microorganisms is crucial in determining plant fitness, and can foster improved tolerance to ...

Candidate Gene Testing in Clinical Cohort Studies with Multiplexed Genotyping and Mass Spectrometry

Complex diseases are often underpinned by multiple common genetic variants that contribute to disease susceptibility. Here, we describe a cost-effective tag single nucleotide polymorphism (SNP) approach using a multiplexed genotyping assay with mass spectrometry, to investigate gene pathway associations in clinical cohorts. We investigate the food allergy candidate locus Interleukin13 (IL13) as an example. This method efficiently maximizes the coverage by taking advantage of shared linkage disequilibrium (LD) within a region. Selected LD SNPs are then designed into a multiplexed assay, enabling up to 40 different SNPs to be analyzed simultaneously, boosting cost-effectiveness. Polymerase chain reaction (PCR) is used to amplify the target loci, followed by single nucleotide extension, and the amplicons are then measured using matrix-assisted laser desorption/ionization-time of flight(MALDI-TOF) mass spectrometry. The raw output is analyzed with the genotype calling software, using stringent quality control definitions and cut-offs, and high probability genotypes are determined and output for data analysis.

Identification of genetic variants contributing to complex human disease allows us to identify novel mechanisms. Here, we demonstrate a multiplex genotyping approach to candidate genes or gene pathway analysis that maximizes the coverage at low cost and is amenable to cohort-based studies.

Complex diseases are often underpinned by multiple common genetic variants that contribute to disease susceptibility. Here, we describe a cost-effective ...