Investigation Title Brugia pahangi mature vs immature microfilariae Comment[Submitted Name] Brugia pahangi mature vs immature microfilariae Experimental Design transcription profiling by array Experimental Design Term Source REF EFO Comment[SecondaryAccession] GSE14939 Comment[ArrayExpressReleaseDate] 2010-05-22 Comment[AEMIAMESCORE] 3 Comment[Publication DOI] 10.1007/s00436-009-1655-y Comment[ArrayExpressAccession] E-GEOD-14939 Comment[MAGETAB TimeStamp_Version] 2010-08-01 20:21:13 Last Changed Rev: 13058 Experimental Factor Name DEVELOPMENTAL STAGE Experimental Factor Type developmental stage Experimental Factor Term Source REF Person Last Name Mayhew McDermott Christensen Griffiths Fuchs Erickson Zink Michalski Person First Name George Colleen Bruce Kathryn Jeremy Sara Rebecca Michelle Person Mid Initials Person Email griffiths.kathryn@gmail.com Person Phone Person Fax Person Address Biology and Microbiology , University of Wisconsin Oshkosh , 800 Algoma Blvd, Oshkosh , WI , USA Person Affiliation University of Wisconsin Oshkosh Person Roles submitter Person Roles Term Source REF The MGED Ontology Quality Control Type Quality Control Term Source REF Replicate Type Replicate Term Source REF Normalization Type Normalization Term Source REF Date of Experiment Public Release Date 2010-05-22 PubMed ID 19894065 Publication DOI 19894065 Publication Author List Griffiths KG, Mayhew GF, Zink RL, Erickson SM, Fuchs JF, McDermott CM, Christensen BM, Michalski ML Publication Title Use of microarray hybridization to identify Brugia genes involved in mosquito infectivity. Publication Status journal_article Publication Status Term Source REF The MGED Ontology Experiment Description Filarial nematodes are arthropod-borne nematodes that cause a variety of economically important diseases such as onchocerciasis (river blindness), lymphatic filariasis, and heartworm disease. The most widespread filarial disease of humans is lymphatic filariasis, caused by worms in the genera Wuchereria and Brugia. Lymphatic filariasis is an economic and social burden in endemic countries and affects approximately 119 million people worldwide (Michael, 1997). In humans, the worms live in and block the lymph vessels, causing improper flow of lymph, and inflammation of the lymphatic system. The symptoms are fever, swollen limbs and genitals, generalized malaise, and can progress to a debilitating condition known as elephantiasis This research focuses on the transmission of these worms to the disseminating mosquito host, and it is based on the interesting observation that mf must be at least 7 days old to successfully infect the mosquito (de Hollanda, 1982). Newborn mf that have not ‘matured’ cannot successfully penetrate the midgut of the mosquito, and subsequently cannot develop to the L3 stage (Fuhrman, 1987). Previous work done by another group 20 years ago suggests that the molecular makeup of the worm surface changes during this maturation process (Furman, 1983 a and b). We used microarray analysis to characterize changes in gene expression that take place during the mf maturation process. Understanding the gene expression changes that occur as the mf mature will allow us to understand the nature of the philological transition that allows mf to move from the human to the mosquito host. With this information in hand, we can eventually identify parasite molecules that could be targeted to either stop parasite reproduction or prevent transmission of the mf to the mosquito. This would stop parasite transmission in endemic areas. Brugia pahangi mature mf (30 days and older) RNA was compared to immature mf (3 days and younger). Three biological replicates were performed each with two technical replicates Protocol Name P-GSE14939-10 P-GSE14939-2 P-GSE14939-3 P-GSE14939-11 P-GSE14939-4 P-GSE14939-12 P-GSE14939-5 P-GSE14939-13 P-GSE14939-6 P-GSE14939-14 P-GSE14939-8 P-GSE14939-15 P-GSE14939-9 P-GSE14939-1 Protocol Type specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action nucleic_acid_extraction nucleic_acid_extraction labeling labeling hybridization hybridization image_aquisition image_aquisition feature_extraction feature_extraction bioassay_data_transformation Protocol Description Male jirds (Meriones unguiculatus) that had patent intraperitoneal infections for a minimum of 30 days were sacrificed and worms collected by peritoneal flush with sterile, prewarmed RPMI-1640 medium (Lonza, Walkersville, MD USA). Adults and mature mf were maintained in 37°C in RPMI containing 100 μg/mL penicillin and 100 μg/mL streptomycin (Cambrex, Rockland, ME USA). Equivalent numbers of adult worms were randomly assigned to groups and surgically transplanted into the peritoneum of uninfected jirds. To reduce damage due to excess handling, worms were not enumerated. Peritoneal flushes were microscopically examined in small volume increments (2-4 mL) to remove contaminating adult worms and debris, and mature (>30 day old) mf isolated by centrifugation through a Histopaque 1083 (Sigma Chemical Co., St. Louis, MO USA) gradient (Chandrashekar 1984). To collect immature mf, gerbils with transplanted adults were sacrificed after two days, and immature mf (<3 days old) were collected and purified as above. Mf pellets were resuspended in 250 ul of sterile phosphate buffered saline, flash frozen and stored at -80° C. B. pahangi transfers and harvests were performed in triplicate to generate three biological replicates for microarray hybridization, B. malayi transfers and harvests were performed in duplicate. Generation and collection of Brugia mf Male jirds (Meriones unguiculatus) that had patent intraperitoneal infections for a minimum of 30 days were sacrificed and worms collected by peritoneal flush with sterile, prewarmed RPMI-1640 medium (Lonza, Walkersville, MD USA). Adults and mature mf were maintained in 37°C in RPMI containing 100 μg/mL penicillin and 100 μg/mL streptomycin (Cambrex, Rockland, ME USA). Equivalent numbers of adult worms were randomly assigned to groups and surgically transplanted into the peritoneum of uninfected jirds. To reduce damage due to excess handling, worms were not enumerated. Peritoneal flushes were microscopically examined in small volume increments (2-4 mL) to remove contaminating adult worms and debris, and mature (>30 day old) mf isolated by centrifugation through a Histopaque 1083 (Sigma Chemical Co., St. Louis, MO USA) gradient (Chandrashekar 1984). To collect immature mf, gerbils with transplanted adults were sacrificed after two days, and immature mf (<3 days old) were collected and purified as above. Mf pellets were resuspended in 250 ul of sterile phosphate buffered saline, flash frozen and stored at -80° C. B. pahangi transfers and harvests were performed in triplicate to generate three biological replicates for microarray hybridization, B. malayi transfers and harvests were performed in duplicate. Total mf RNA was isolated by organic extraction with Trizol LS (Invitrogen, Carlsbad, CA USA), followed by column purification using Ambion RNAqueous-Micro® Kit (Applied Biosystems, Foster City, CA USA). RNA integrity was confirmed visually by agarose gel electrophoresis (data not shown), and purity and concentration determined spectrophotometrically (NanoDrop ND-1000, Thermo Fisher Scientific Wilmington, DE USA) and stored at -80° C. Samples were lyophilized under vacuum for transport to the Washington University Genome Sequencing Center, where RNA quality was verified using Agilent 2100 bioanalyzer (Agilent Technologies) Total mf RNA was isolated by organic extraction with Trizol LS (Invitrogen, Carlsbad, CA USA), followed by column purification using Ambion RNAqueous-Micro® Kit (Applied Biosystems, Foster City, CA USA). RNA integrity was confirmed visually by agarose gel electrophoresis (data not shown), and purity and concentration determined spectrophotometrically (NanoDrop ND-1000, Thermo Fisher Scientific Wilmington, DE USA) and stored at -80° C. Samples were lyophilized under vacuum for transport to the Washington University Genome Sequencing Center, where RNA quality was verified using Agilent 2100 bioanalyzer (Agilent Technologies) First strand cDNA is generated by oligo-dT primed reverse transcription (Superscript II; Invitrogen) utilizing the 3DNA Array 900 kit (Genisphere). Modified oligo-dT primers are utilized in which a fluorophore/dendrimer specific oligo sequence is attached to the 5’ end of the dT primer. For cDNA synthesis, 1ul of fluorophore specific oligo-dT primer is added to 2ug of total RNA and the solution is incubated at 80C for 5 minutes then cooled on ice for 2 minutes. To each sample are added RNase inhibitor (Superase-In; Ambion) (0.5ul), 5X first strand buffer (2ul), dNTP mix (10mM each dATP, dCTP, dGTP, and dTTP) (0.5ul), 0.1M DTT (1ul), and Superscript II RNase H- Reverse Transcriptase (0.5ul). Reverse transcription is carried out at 42C for 2 hours. The reaction is terminated by adding 1.0M NaOH/ 100mM EDTA (1.0ul) and incubation at 65C for 10 minutes then neutralized with 2M Tris-HCL, pH 7.5 (1.2ul). For RNA expression level comparison, samples are paired. First strand cDNA is generated by oligo-dT primed reverse transcription (Superscript II; Invitrogen) utilizing the 3DNA Array 900 kit (Genisphere). Modified oligo-dT primers are utilized in which a fluorophore/dendrimer specific oligo sequence is attached to the 5’ end of the dT primer. For cDNA synthesis, 1ul of fluorophore specific oligo-dT primer is added to 2ug of total RNA and the solution is incubated at 80C for 5 minutes then cooled on ice for 2 minutes. To each sample are added RNase inhibitor (Superase-In; Ambion) (0.5ul), 5X first strand buffer (2ul), dNTP mix (10mM each dATP, dCTP, dGTP, and dTTP) (0.5ul), 0.1M DTT (1ul), and Superscript II RNase H- Reverse Transcriptase (0.5ul). Reverse transcription is carried out at 42C for 2 hours. The reaction is terminated by adding 1.0M NaOH/ 100mM EDTA (1.0ul) and incubation at 65C for 10 minutes then neutralized with 2M Tris-HCL, pH 7.5 (1.2ul). For RNA expression level comparison, samples are paired. Each sample pair (~24ul) is resuspended in formamide-based hybridization buffer (vial 7-Genisphere) (26ul), and Array 50dT blocker (Genisphere) (2ul). Two hybridizations are carried out in a sequential manner. The primary hybridization is performed by adding 48ul of sample to the microarray under a supported glass coverslip (Erie Scientific) at 43C for 16-20 hours at high humidity in the dark. Prior to the secondary hybridization, slides are gently submerged into 2X SSC, 0.2% SDS (at 43C) for 11 min., transferred to 2X SSC (at room temp.) for 11 min., transferred to 0.2X SSC (at room temp.) for 11 min., and then spun dry by centrifugation. Secondary hybridization is carried out using the complimentary capture reagents provided in the 3DNA Array 900 kit (Genisphere). For each reaction, the following are added: 3DNA capture reagent with Cy3 (2.5ul), 3DNA capture reagent with Cy5 (2.5ul), SDS-based hybridization buffer (vial 6-Genisphere) (26ul), and nuclease-free water (21ul). The secondary hybridization solution is incubated in the dark at 80C for 10 min. then 50C for 15 min. Hybridization is performed by adding 48ul secondary hybridization solution to the slide under a supported glass coverslip at 65C for 4 hr at high humidity in the dark. At hybridization termination, arrays are gently submerged into 2X SSC, 0.2% SDS (at 65C) for 11 min., transferred to 2X SSC (at room temp.) for 11 min., transferred to 0.2X SSC (at room temp.) for 11 min., and then spun dry by centrifugation. Hybridization Slides are scanned on a Perkin Elmer ScanArray Express HT scanner to detect Cy3 and Cy5 fluorescence. Laser power is kept constant for Cy3/Cy5 scans and PMT is varied for each experiment based on optimal signal intensity with lowest possible background fluorescence. A low pmt setting scan is also performed to recover signal from saturated elements. Gridding and analysis of images is performed using ScanArray v3.0 (Perkin Elmer). Slides are scanned on a Perkin Elmer ScanArray Express HT scanner to detect Cy3 and Cy5 fluorescence. Laser power is kept constant for Cy3/Cy5 scans and PMT is varied for each experiment based on optimal signal intensity with lowest possible background fluorescence. A low pmt setting scan is also performed to recover signal from saturated elements. Gridding and analysis of images is performed using ScanArray v3.0 (Perkin Elmer). Slides were scanned on a Perkin Elmer ScanArray Express HT scanner to detect Cy3 and Cy5 fluorescence as described by Li et al. B. pahangi data were analyzed by volcano plots (Bartholomay 2007). GeneSpring software (Agilent Technologies, Santa Clara, CA, USA) was used to identify differentially abundant transcripts using a 95% confidence interval over twofold values. To reduce false positives due to only two biological replicates in the B. malayi study, results were filtered using Benjamini and Hochberg false discovery rate (Benjamini 2001). Slides were scanned on a Perkin Elmer ScanArray Express HT scanner to detect Cy3 and Cy5 fluorescence as described by Li et al. B. pahangi data were analyzed by volcano plots (Bartholomay 2007). GeneSpring software (Agilent Technologies, Santa Clara, CA, USA) was used to identify differentially abundant transcripts using a 95% confidence interval over twofold values. To reduce false positives due to only two biological replicates in the B. malayi study, results were filtered using Benjamini and Hochberg false discovery rate (Benjamini 2001). ID_REF =
VALUE = normalized ratio Log2 (mature/immature)
t-test p-value = Protocol Parameters Protocol Hardware Protocol Software Protocol Contact Protocol Term Source REF The MGED Ontology The MGED Ontology The MGED Ontology SDRF File E-GEOD-14939.sdrf.txt Term Source Name The MGED Ontology ArrayExpress EFO The MGED Ontology Term Source File http://mged.sourceforge.net/ontologies/MGEDontology.php http://www.ebi.ac.uk/arrayexpress http://www.ebi.ac.uk/efo/ http://mged.sourceforge.net/ontologies/MGEDontology.php Term Source Version