Name: Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR (Video) | JoVE Journal
Uploaded: 2011-07-30T12:00:00.000+00:00
Duration: 10 min 27 s
Description: Learn microarray analysis and real-time PCR to investigate gene expression profiles in C. elegans. This computational method identifies candidate genes and validates findings using bioinformatic tools.

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5 minutes

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/pubmed?term=((hVAPB%2C+the+causative+gene+of+a+heterogeneous+group+of+motor+neuron+diseases+in+humans%2C+is+functionally+interchangeable+with+its+Drosophila+homologue+DVAP-33A+at+the+Neuromuscular+Junction%5BTitle%5D)+AND+%22Hum+Mol+Genet%22%5BJournal%5D)">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/pubmed?term=((The+Amyotrophic+Lateral+Sclerosis+8+protein+VAPB+is+cleaved%2C+secreted%2C+and+acts+as+a+ligand+for+Eph+receptors%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">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/pubmed?term=((Assembly+of+Active+Zone+Precursor+Vesicles%5BTitle%5D)+AND+%22Journal+of+Biological+Chemistry%22%5BJournal%5D)">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/pubmed?term=((Yeast+VSM1+Encodes+a+v-SNARE+Binding+Protein+That+May+Act+as+a+Negative+Regulator+of+Constitutive+Exocytosis%5BTitle%5D)+AND+%22Molecular+and+Cellular+Biology%22%5BJournal%5D)">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>Viñuela, A., Snoek, L., Riksen, J., Kammenga, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genome-Wide+Gene+Expression+Analysis+in+Response+to+Organophosphorous+Pesticide+Chlorpyrifos+and+Diazinon+in+C.+elegans%5BTitle%5D)+AND+%22PloS+one%22%5BJournal%5D)">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/pubmed?term=((elegans+sym-1+is+downstream+target+of+the+hunchback-like-1+developmental+timing+transcription+factor%5BTitle%5D)+AND+%22Cell+Cycle%22%5BJournal%5D)">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/pubmed?term=((Genome-wide+analysis+of+mRNA+targets+for+Caenorhabditis+elegans+FBF%2C+a+conserved+stem+cell+regulator%5BTitle%5D)+AND+%22PNAS%22%5BJournal%5D)">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/pubmed?term=((Homologs+of+genes+expressed+in+Caenorhabditis+elegans+GABAergic+neurons+are+also+found+in+the+developing+mouse+forebrain%5BTitle%5D)+AND+%22Neural+Development%22%5BJournal%5D)">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><tbody><tr><td>RNA Later</td><td>Ambion</td><td>AM7020</td><td></td></tr><tr><td>Molecular Grinding Resin</td><td>G-Biosciences</td><td>786-138</td><td></td></tr><tr><td>RNeasy Mini Kit</td><td>Qiagen</td><td>74104</td><td></td></tr><tr><td>Rnase-free Agarose LE</td><td>Ambion</td><td>AM9040</td><td></td></tr><tr><td>NorthernMax Gly 10X Gel Prep/Running Buffer</td><td>Ambion</td><td>8678</td><td></td></tr><tr><td>RNA Millenium Marker</td><td>Ambion</td><td>AM7150</td><td></td></tr><tr><td>Glyoxal Sample Loading Dye</td><td>Ambion</td><td>8551</td><td></td></tr><tr><td>DNase set</td><td>Qiagen</td><td>79254</td><td></td></tr><tr><td>RNeasy MinElute Kit</td><td>Qiagen</td><td>74204</td><td></td></tr><tr><td>RT Enzyme and 5X Reaction Buffer</td><td>Genisphere</td><td>RT300320</td><td></td></tr><tr><td>Array 350</td><td>Genisphere</td><td>W300180</td><td></td></tr><tr><td>QIAquick PCR Purification Kit</td><td>Qiagen</td><td>28104</td><td></td></tr><tr><td>20X SSC</td><td>VWR international</td><td>82021-4846</td><td></td></tr><tr><td>Sheared Salmon Sperm DNA</td><td>Ambion</td><td>AM9680</td><td></td></tr><tr><td>SDS Solution</td><td>Promega Corp.</td><td>V6551</td><td></td></tr><tr><td>DTT EM</td><td>VWR international</td><td>3860</td><td></td></tr><tr><td>iScript cDNA Synthesis Kit</td><td>Bio-Rad</td><td>170-8890</td><td></td></tr><tr><td>iQ SYBR Green Supermix</td><td>Bio-Rad</td><td>170-8880</td><td></td></tr></tbody></table>

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

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.

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

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, ...

The overall goal of this procedure is to investigate gene expression profiles underlying numerous phenotypes or metabolic pathways. This is accomplished by first isolating mRNA from two contrasting nematode strains. The second step of the procedure is to synthesize CD NA containing either S SI three or SFI capture sequences and hybridize them to a microarray.The third step of the procedure is to hybridize the microarray with anti capture sequences containing S SI three and SCIFI fluoro fours. The final step of the procedure is to scan the microarray and analyze data using bioinformatic tools such as the magic tool software. Ultimately, candidate genes mediating the biological phenomenon under investigation are identified and can be validated and quantified by alternative techniques like real-time PCR.Generally, individuals new to this method will struggle because spotted nucleotides are not visible to the user, so it is difficult to overlay a homogenous hybridization mixture onto the 23, 000 plus. Uniquely printed nucleotides Begin by collecting RNA from healthy synchronized nematodes. First, they're washed resuspended in 15 milliliter tubes and pelleted worm pellets are resuspended in seven milliliters of 0.1 molar sodium chloride and seven milliliters of ice cold, 60%weight per volume sucrose.After a 15 minute incubation on ice, the worms are pelleted again. Now bacteria free worms will swim up, are collected, washed in RNAs, free water and pelleted. Again, the clean, loosely compacted pellet is transferred to a micro centrifuge tube spun down at maximum speed for 30 seconds.Aspirated and stored in 10 volumes of RNA later at four degree Celsius until ready to proceed. To prepare total RNA samples, centrifuge the prepared pellets at maximum speed, discard the supernat and add a pinch of molecular grinding resin to the pellet. Then freeze the mix using liquid nitrogen using a pestle.Grind the frozen worm suspension into a fine powder ad, liquid nitrogen as needed to keep the powder cold after grinding. Put the extract on ice for five minutes. After five minutes, mix the extract with 700 microliters of R-L-T-B-M-E and 472 microliters of 100%ethanol total.RNA can now be isolated using a commercially available kit to a final volume of 60 microliters of isolated RNA per biological sample. Before proceeding, remove potential genomic DNA contamination by treating with DNAS one, followed by use of a commercially available RNA cleanup kit. Complete the kit by alluding to a 60 microliter volume.Finally, determine the RNA concentration and evaluate the integrity of the RNA by treatment with glyoxal sample loading, dye and gel analysis prior to microarray hybridization Using conventional methods purified RNA is reversed transcribed into complimentary DNA, which is purified from the degraded template with a commercially available kit and alluded to a volume of 60 microliters. Begin the microarray hybridization by blocking sea elegance. Microarray slides with sonicated salmon sperm DNA After an hour, tip the blocked slides in double distilled water and spin.Dry the slides in 50 milliliter conical tubes padded by a kim wipe. Now prepare a two x form amide based hybridization buffer. First, warm it to 55 degrees Celsius for 10 minutes to fully dissolve its crystals.Then centrifuge it for one minute at 10, 000 gs. Next, mix 25 microliters of CD NA with 25 microliters of hybridization buffer, and flick the mixture gently. Now perform a quick spin of the mix and incubate it at 80 degrees Celsius for 10 minutes.After the incubation, carefully pipette the entire CDNA sample onto the microarray slide without touching the slide. To uniformly spread the sample onto the microarray slide. Gently lower a cover slip onto the slide using a syringe needle before the cover slip is completely lowered, pull back up and lower with the needle again allowing the cover slip to gently fall into place.This technique minimizes the formation of air bubbles. Now place the slide horizontally in a 50 milliliter conical tube below the slide at 50 microliters of double distilled water and allow it to incubate overnight at 37 degrees Celsius the next day. A second hybridization is performed briefly after the slide is washed.Multiple times in SSC, the second buffer, which contains light sensitive capture reagents and an Antifa reagent is applied using the same technique followed by a short incubation, more washes and drying. The slide can then be scanned. Images can be analyzed using the free software with open source magic tool.Briefly under the project tab, create and save a new project under the build expression profile tab, select load image pair and select the red image file as red and the green image file as green. Then select the build expression profile tab. Choose load gene list to upload a C elegance gene list file obtained from the GCAT website to address and grid the microarray image.Select the build expression profile tab and create edit grid option. Now edit the grid. Set up dialogue box using 48 for the number of grids, 22 rows and 22 columns for each grid and choosing to number spots from left to right and top to bottom.Increase the percent contrast change and zoom in on the grid until the individual spots are easily distinguishable. Create the grids by selecting set top left spot on the left hand panel and then clicking the center of the top left spot, followed by the top right spot and bottom row on grid one. Repeat this gridding procedure for each of the 48 grids proceeding left to right and top to bottom.Then save by selecting the file tab and save current grid as once saved. Select the finished tab to eliminate any unrelated spots from the analysis. Open the build expression profile tab and under the addressing gridding option, select spot flagging.Now flag any streak spots or background fluorescence and save by selecting save current flags. As under the file tab, the flagged files should be segmented. Lastly, analyze genes induced or repressed by a specified factor by selecting explore under the expression tab.In the exploring dialogue box, set the fine genes matching criteria. The number of twofold increases or decreases in expression. Intensity is known as x and the value of X is the criteria for expression change.X is input under max value greater than X for induced genes and min value less than x for repressed genes. In this experiment, a dye swap control is performed the two independent total RNA isolations from stage three and stage four larvae are hybridized as either green mutant versus red wild type, whereas red mutant versus green wild type to confirm gene expression changes make three independent total RNA isolations using the same procedure synthesize CDNA with a commercially available kit, and for each CD NA sample perform real-time PCR and triplicate using three housekeeping genes as controls. For demonstration, the following results examine gene expression induced in VSM one.AK 1468 mutants a nematode strain characterized by enhanced synaptic prior to performing microarray analysis, the quality of the extracted RNA is checked using gel electrophoresis. The presence of two intact ribosomal subunits before and after dase one treatment is indicative of a good RNA sample in the microarray wild type CDNA is labeled red and VSM one mutant CD NA is labeled green. In this one of the 48 grids analyzed per microarray, each small square represents a single printed oligonucleotide in each array, each unique oligonucleotide is represented.Once microarray analysis of transcripts isolated from stage four larvae shows that genes coding for the major sperm proteins or MSP are induced in VSM one mutants microarray analysis of stage three larvae also reveals expression changes within the same gene family in the mutant.Testing. Predictions from the microarray by realtime PCR analysis reveals that one member of the M ms P gene family MS P 32, is induced in the VSM one mutants. The data represents the average of three replicates of real-time PCR from the same RNA collection.After watching this video, you should have a good understanding of how to handle bioinformatic tools for genome wide analysis, especially considering there are many open source bioinformatic software programs available to the scientific community.

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