Unknown,Transcriptomics,Genomics,Proteomics

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Schistosoma japonicum gender-associated differentially expressed genes

ABSTRACT: Suppression subtractive hybridization(SSH) libraries of Schistosoma japanicum female and male worms were constructed by using Clontech PCR-selectTM cDNA subtraction kit. S.japonicum cDNA microarrays were fabricated using female and male cDNA clones originating from SSH libraries. female-associated and Male-associated differentially expressed gene clones were obtained, Analysis of gender-associated differentially expressed genes helps to determine which genes are important to sexual maturation of schistosome and better understand schistosome biology and host-parasite relationship, facilitate the discovery of novel gene products that could represent targets for the development of new drugs and vaccines to control chitosomiasis. Keywords: gender-associated Four Samples were analyzed about Schistosoma japonicum gender-associated differentially expressed genes

ORGANISM(S): Schistosoma japonicum

SUBMITTER: Yanxun Xia

PROVIDER: E-GEOD-3641 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:We used complementary DNA microarray to analyze the gene expression profiles in different proglottids of Moniezia expansa. A total of 4056 sequences including full length and partial complementary DNAs representing novel, known, and control genes were analyzed. We utilized cDNA array for detection of gene expression profiles of different proglottids of M.expansa. The present study provides some interesting data to better understanding the mechanisms of procreation and may suggest some potential target molecules for a more effective treatment on verminosis. 1.Construction of microarray By clustering the 2,642 sequences from the cDNA library, 1,078 unigenes of M.expansa, including full-length and partial cDNAs representing novel or known genes, were obtained for array construction. The negative control spots of non-tapeworm origin in the chip were the rice genes (48 spots). In addition, the array included 9 known genes(54 spots) as positive control genes that were provided by our library and 24 empty spots. Unigenes and all control genes were cloned into plasmid vector. The DZH (TC) chips were constructed by United Gene Holdings Limited of China (Shanghai, China) according to the method described by Li et al. The cDNA inserts were amplified by use of the polymerase chain reaction (PCR) using universal primers to plasmid vector sequences and were then purified. All PCR products were examined by agarose gel electrophoresis to ensure the quality and the identity of the amplified clones as expected. Then the amplified PCR products were dissolved in a buffer containing 3ÃSSC solution. The solution with amplified PCR products were spotted onto glass slides (model DZH-TC) using a Cartesian PixSys 7500 motion control robot (Cartesian Technologies, Irvine, CA., USA) fitted with ChipMaker Micro-Spotting Technology (TeleChem International, Sunnyvale, CA., USA). The glass slides were then hydrated for 2 h in 70% humidity, dried for 0.5 h at room temperature, and UV crosslinked (65 mj/cm). They were further processed at room temperature by soaking in 0.2% sodium dodecyl sulfate (SDS) for 10 min, distilled H2O for 10 min, and 0.2% sodium borohydride (NaBH4) for 10 min. The slides were dried again and ready for use. 2.RNA preparation and probe labeling Total RNAs were isolated from 6 M.expansas with scolex-neck proglottids, immature proglottids, mature proglottids and gravid proglottids. Tissue samples were ground into a fine powder in a 10 cm ceramic mortar (RNase-free) and were then homogenized in TRIzol (Biostar, Shanghai, China). After centrifugation, the supernatant was separated from the organic phase and was extracted in an equal volume of chloroform. The aqueous phase was then precipitated by an equal volume of isopropanol at 4Â°C, centrifuged to pellet the RNA and dissolved in Milli-Q H2O. The fluorescent cDNA probes were prepared through reverse transcription with Cy3- or Cy5-deoxy UTP (Amersham Pharmacia Biotech, Piscataway, NJ, USA) as follows: 5 Î¼g of oligo(dT18) was added and annealed to 3Î¼g of mRNA by heating the mixture to 70ËC for 10 min, and then chilling it on ice. The final reaction buffer mixture contained dNTPs (200 Î¼M dATP, dCTP and dGTP; 60 Î¼M dTTP; and 60 Î¼M Cy3- or Cy5-dUTP), 2 Î¼l of Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA, USA) and 1Ãreaction buffer 10Î¼l. The reactions were carried out at 42 ÂºC for 2 h. RNA was hydrolyzed by adding 4 Î¼l of 2.5 M NaOH, incubating the mixture at 65 ÂºC for 10 min and then neutralizing it with 4 Î¼l of 2.5 M HCl. The RNA samples from the scolex-neck proglottids were labeled with Cy3-dUTP and those from immature proglottids, mature proglottids and gravid proglottids, respectively, were labeled with Cy5-dUTP. The two color probes were then mixed and diluted to 500 Î¼l with TE, and concentrated using a Microcon YM-30 filter (Millipore, Bedford, MA, USA) to 10 Î¼l. The sample was then vacuum dried. 3.Hybridization The probe was dissolved in 20 ml of hybridization solution (5ÃSSC (0.75M NaCl and 0.075M sodium citrate), 0.4% SDS, 50% formamide). Microarrays were pre-hybridized with a hybridization solution containing 0.5 mg/ml of denatured salmon sperm DNA at 42Â°C for 6 h. Fluorescent probe mixtures were denatured at 95Â°C for 5 min and then applied onto the pre-hybridized chip under a cover glass. Chips were hybridized at 42Â°C for 15-17 h. Next, the hybridized chips were each washed at 60Â°C for 10 min in solutions of 2ÃSSC and 0.2% SDS, 0.1ÃSSC and 0.2% SDS, 0.1ÃSSC and then dried at room temperature. 4.Detection and analysis The chips were scanned with a ScanArray 4000 (GSI Lumonics, Bellerica, MA, USA) at two wavelengths, 635 and 532 nm, to detect emission from both Cy5 and Cy3, respectively. The acquired images were analyzed using GenePix Pro 3.0 software. The intensities of each spot at the two wavelengths represent the quantity of Cy3-dUTP and Cy5-dUTP. Ratios of Cy5 to Cy3 were computed using the GenePix Pro 3.0 median of ratio method. Overall intensities were normalized using the corresponding GenePix default normalization factor. All spots flagged âBadâ or âNot Foundâ by GenePix software were removed from the final data. Only genes with raw intensity values for both Cy3 and Cy5 of >200 were chosen for differential analysis. Genes were identified as differentially expressed if the ratio was >2 or <0.5, or the absolute value of base 2 logarithm of the ratio was >1 or < -1. mature proglottids, immature proglottids, gravid proglottids vs common reference scolex-neck proglottids tissue. 2 biological replicates for each experiments.

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Schistosoma japonicum gender-associated differentially expressed genes

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