Name: determining genetic expression profiles in c. elegans using microarray and real-time pcr
Uploaded: 2011-07-30T12:00:00
Duration: 10 min 27 s
Description: Watch this Scientific Journal Video about Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR at JoVE.com

This work was performed by undergraduate students at Southwestern Oklahoma State University, Weatherford OK. This work was supported by NSF RUI-0963258, NIH OK-INBRE 2P20RR016478, GCAT and OCAST HR09-137S.
The vsm1(ok1468) mutant was isolated by the Oklahoma Gene Knockout Consortium. 
The authors thank Dan Stefanovic and Tanner Wheeler for their valuable help during the project execution.

<ol>
	<li>Chai, A., Withers, J., Koh, Y. H., Parry, K., Bao, H., Zhang, B., Budnik, V., Pennetta, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=hVAPB,+the+causative+gene+of+a+heterogeneous+group+of+motor+neuron+diseases+in+humans,+is+functionally+interchangeable+with+its+Drosophila+homologue+DVAP-33A+at+the+Neuromuscular+Junction.">hVAPB, the causative gene of a heterogeneous group of motor neuron diseases in humans, is functionally interchangeable with its Drosophila homologue DVAP-33A at the Neuromuscular Junction.</a> Hum Mol Genet. 17, 266-280 (2008).</li><li>Tsuda, H., Han, S. M., Yang, Y., Tong, C., Lin, Y. Q., Mohan, K., Haueter, C., Zoghbi, A., Harati, Y., Kwan, J., Miller, M., Bellen, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Amyotrophic+Lateral+Sclerosis+8+protein+VAPB+is+cleaved,+secreted,+and+acts+as+a+ligand+for+Eph+receptors.">The Amyotrophic Lateral Sclerosis 8 protein VAPB is cleaved, secreted, and acts as a ligand for Eph receptors.</a> Cell. 133, 963-977 (2008).</li><li>Dresbach, T., Torres, V., Wittenmayer, N., Altrock, W. D., Zamorano, P., Zuschratter, W., Nawrotzki, R., Ziv, N. E., Garner, C. C., Gundelfinger, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assembly+of+Active+Zone+Precursor+Vesicles.">Assembly of Active Zone Precursor Vesicles.</a> Journal of Biological Chemistry. 281, 6038-6047 (2005).</li><li>Lustgarten, V., Gerst, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yeast+VSM1+Encodes+a+v-SNARE+Binding+Protein+That+May+Act+as+a+Negative+Regulator+of+Constitutive+Exocytosis.">Yeast VSM1 Encodes a v-SNARE Binding Protein That May Act as a Negative Regulator of Constitutive Exocytosis.</a> Molecular and Cellular Biology. 19, 4480-4494 (1999).</li><li>Vi&#241;uela, A., Snoek, L., Riksen, J., Kammenga, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-Wide+Gene+Expression+Analysis+in+Response+to+Organophosphorous+Pesticide+Chlorpyrifos+and+Diazinon+in+C.+elegans.">Genome-Wide Gene Expression Analysis in Response to Organophosphorous Pesticide Chlorpyrifos and Diazinon in C. elegans.</a> PloS one. 5, 1-8 (2010).</li><li>Niwa, R., Hada, K., Moliyama, K., Ohniwa, R., Tan, Y., Olsson-Carter, K., Chi, W., Reinke, V., Slack, F. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=elegans+sym-1+is+downstream+target+of+the+hunchback-like-1+developmental+timing+transcription+factor.">elegans sym-1 is downstream target of the hunchback-like-1 developmental timing transcription factor.</a> Cell Cycle. 8, 4147-4154 (2009).</li><li>Kershner, A., Kimble, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+analysis+of+mRNA+targets+for+Caenorhabditis+elegans+FBF,+a+conserved+stem+cell+regulator.">Genome-wide analysis of mRNA targets for Caenorhabditis elegans FBF, a conserved stem cell regulator.</a> PNAS. 107, 3963-3941 (2010).</li><li>Hammock, E., Eagleson, K., Barlow, S., Earls, L., Miller, D., Levitt, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homologs+of+genes+expressed+in+Caenorhabditis+elegans+GABAergic+neurons+are+also+found+in+the+developing+mouse+forebrain.">Homologs of genes expressed in Caenorhabditis elegans GABAergic neurons are also found in the developing mouse forebrain.</a> Neural Development. 5, 1-14 (2010).</li></ol>

No conflicts of interest declared.

In this study we developed an easy, user friendly protocol that can be used to determined gene expression profiles underlying various biological phenomena. In this protocol, total RNA was first isolated and treated using DNase I. Our method of RNA isolation has been dramatically simplified by omitting phenol extractions and using affinity resins for clean up 5,6. Briefly, C. elegans cuticule and cell membranes were cracked-open by freezing nematodes with liquid nitrogen and grinding with molecular resin. Next, extracted RNA was treated with DNase I before cDNA synthesis. The later step was added to minimize unspecific hybridizations; RNA samples contaminated with genomic DNA could introduce interference and produce many false-positives. Then, wild type and vsm-1(ok1468) mutant cDNAs were tagged with Cy3 and Cy5 capture sequences and hybridized onto C. elegans microarrays obtained through the GCAT program. This system is more sensitive than similar products, and its two-color design decreases the number of arrays needed, resulting in greater time and cost efficiency 7,8. Additionally, this system does not require the specialized equipment of other similar systems; therefore, this procedure could be accomplished in any standard laboratory setting 7. Third, fluorescent hybridized array images were analyzed using the open source software MAGICTool, and gene expression profiles were created. MAGICTool is a user-friendly program that is easily utilized by individuals of all experience levels, from the undergraduate to the post-doctorate. However, optimal performance requires large memory availability. Lastly, candidate genes obtained using microarray analysis were validated with real time PCR. 
Though the genomic approach is an efficient method for studying biological phenomena, there are some limitations one should take into consideration. First, despite the specificity of this microarray protocol, transcripts within a family are indistinguishable from one another if the printed oligonucleotide is taken from conserved sequences. Real time PCR compensates for similarities within a gene family and may even distinguish between specific isoforms of the same gene. However, with real time PCR it is a challenge to find true controls that are expressed equally throughout an organism's lifespan. Because of this, results will be relative. A proteomic approach is one alternative to our technique. Individual proteins may be isolated using 2D gel electrophoresis and identified with mass spectrometry.
Our studies specifically show that many members of the Major Sperm Protein (MSP) family are induced in vsm-1 mutant nematodes with enhanced synaptic density. MSPs are unique to C. elegans and are responsible for oocyte maturation and ovulation. Chai 1 has demonstrated that regulation of presynaptic bouton number and size at the neuromuscular junction in fruit flies is likely controlled by molecules containing Major Sperm Protein domains. Studies by Tsuda and colleagues 2 have demonstrated Major Sperm Protein domains may serve as ligands for Ephrine receptors, triggering receptor tyrosine kinase signaling cascades. Moreover, real time PCR analysis validated and narrowed down microarray results to one member of the msp family, msp-32. Taken together, the work presented here represents a genome-wide analysis of a central neuroscience dilemma as well as the power of undergraduate research in the scientific endeavor.

<table border="1">
	<tbody>	
 <tr>
 <td>Component</td>
 <td>Catalog #</td>
 <td>Company</td>
 </tr>
 <tr>
 <td>RNA Later</td>
 <td>AM7020</td>
 <td>Ambion</td>
 </tr>
 <tr>
 <td>Molecular Grinding Resin</td>
 <td>786-138</td>
 <td>Gbiosciences</td>
 </tr>
 <tr>
 <td>RNeasy Mini Kit</td>
 <td>74104</td>
 <td>Qiagen</td>
 </tr>
 <tr>
 <td>Rnase-free Agarose LE</td>
 <td>AM9040</td>
 <td>Ambion</td>
 </tr>
 <tr>
 <td>NorthernMax Gly 10X Gel Prep/Running Buffer</td>
 <td>8678</td>
 <td>Ambion</td>
 </tr>
 <tr>
 <td>RNA Millenium Marker</td>
 <td>AM7150</td>
 <td>Ambion</td>
 </tr>
 <tr>
 <td>Glyoxal Sample Loading Dye</td>
 <td>8551</td>
 <td>Ambion</td>
 </tr>
 <tr>
 <td>DNase set</td>
 <td>79254</td>
 <td>Qiagen</td>
 </tr>
 <tr>
 <td>RNeasy MinElute Kit</td>
 <td>74204</td>
 <td>Qiagen</td>
 </tr>
 <tr>
 <td>RT Enzyme and 5X Reaction Buffer</td>
 <td>RT300320</td>
 <td>Genisphere</td>
 </tr>
 <tr>
 <td>Array 350</td>
 <td>W300180</td>
 <td>Genisphere</td>
 </tr>
 <tr>
 <td>QIAquick PCR Purification Kit</td>
 <td>28104</td>
 <td>Qiagen</td>
 </tr>
 <tr>
 <td>20X SSC</td>
 <td>82021-4846</td>
 <td>VWR</td>
 </tr>
 <tr>
 <td>Sheared Salmon Sperm DNA</td>
 <td>AM9680</td>
 <td>Ambion</td>
 </tr>
 <tr>
 <td>SDS Solution</td>
 <td>V6551</td>
 <td>Promega</td>
 </tr>
 <tr>
 <td>DTT EM</td>
 <td>3860</td>
 <td>VWR</td>
 </tr>
 <tr>
 <td>iScript cDNA Synthesis Kit</td>
 <td>170-8890</td>
 <td>BioRad</td>
 </tr>
 <tr>
 <td>iQ SYBR Green Supermix</td>
 <td>170-8880</td>
 <td>BioRad</td>
 </tr>
 </tbody>
</table>

determining genetic expression profiles in c. elegans using microarray and real-time pcr

Synapses are composed of a presynaptic active zone in the signaling cell and a postsynaptic terminal in the target cell. In the case of chemical synapses, messages are carried by neurotransmitters released from presynaptic terminals and received by receptors on postsynaptic cells. Our previous research in Caenorhabditis elegans has shown that VSM-1 negatively regulates exocytosis. Additionally, analysis of synapses in vsm-1 mutants showed that animals lacking a fully functional VSM-1 have increased synaptic connectivity. Based on these preliminary findings, we hypothesized that C. elegans VSM-1 may play a crucial role in synaptogenesis. To test this hypothesis, double-labeled microarray analysis was performed, and gene expression profiles were determined. First, total RNA was isolated, reversely transcribed to cDNA, and hybridized to the DNA microarrays. Then, in-silico analysis of fluorescent probe hybridization revealed significant induction of many genes coding for members of the major sperm protein family (MSP) in mutants with enhanced synaptogenesis. MSPs are the major component of sperm in C. elegans and appear to signal nematode oocyte maturation and ovulation . In fruit flies, Chai and colleagues 1 demonstrated that MSP-like molecules regulate presynaptic bouton number and size at the neuromuscular junction. Moreover, analysis performed by Tsuda and coworkers 2 suggested that MSPs may act as ligands for Eph receptors and trigger receptor tyrosine kinase signaling cascades. Lastly, real time PCR analysis corroborated that the gene coding for MSP-32 is induced in vsm-1(ok1468) mutants. Taken together, research performed by our laboratory has shown that vsm-1 mutants have a significant increase in synaptic density, which could be mediated by MSP-32 signaling.

Watch this Scientific Journal Video about Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR at JoVE.com

Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR

Microarray analysis was conducted to determine genetic expression profiles in C. elegans, and real-time PCR was used to validate and quantify microarray data.

Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR

Synapses are composed of a presynaptic active zone in the signaling cell and a postsynaptic terminal in the target cell. In the case of chemical synapses, ...

determining-genetic-expression-profiles-c-elegans-using-microarray

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