Project description:Subconfluent AG01518 fibroblasts were starved for 16 hours in the presence of 0.1% BSA, and then stimulated for 1, 4, 10 or 24 hours with PDGF-BB (10 ng/ml in starvation medium), or left untreated for the same periods of time. Total RNA was isolated using the RNeasy kit (Qiagen). The RNA quality was checked by formaldehyde agarose gel electrophoresis (RNeasy protocol, Qiagen). Total RNA (40 mg) from cells treated for a given period of time with PDGF or control medium were labelled in reverse transcription reactions (Superscript II kit, Invitrogen) with dCTP-Cy5 and dCTP-Cy3, respectively (Amersham). In every second replicate experiment the fluorescent deoxynucleotides were swapped. Purified cDNA probes labelled with Cy3 and Cy5 were mixed per pair, and hybridized to cDNA microarray chips (Sanger Institute H. sapiens 10K array, Hver1.2.1) from the Sanger Institute/LICR/CRUK Consortium (see http://www.sanger.ac.uk/Projects/Microarrays/ for details and hybridization protocols). For each time point, we performed at least four independent hybridizations, and used at least two different batches of RNA. Chips were scanned in a Perkin Elmer/GSI Lumonics ScanArray 4000 scanner and spot intensities were measured using the QuantArray software (histogram method with background subtraction). Normalization and statistical analysis of the quadruplicate data sets were performed using GeneSpring 5.0 analysis software (Silicon Genetics). A Lowess non-linear normalization was applied and the median of the ratio distribution for each array was set to 1. Regulated spots were selected based on the average ratio values > 1.750 for up-regulated genes and < 0.571 for down-regulated genes. In addition, we considered only genes that were significantly regulated (t-test, p<0.05) based on replicate hybridizations (global error model, GeneSpring). For all features selected using this protocol, the signal was significantly above the background, indicating that the expression of these genes was detectable. Finally, Hver1.2.1 microarrays contain replicate spots (1 to 6) corresponding to the same gene. Genes represented by spots that were not regulated in a similar manner were discarded. We show the average ratio of one representative spot for each regulated gene, with standard error calculated from multiple hybridizations and with the annotation provided by the microarray facility (Hver1.2.1_NCBI33).

Project description:Subconfluent AG01518 fibroblasts were starved for 16 hours in the presence of 0.1% BSA, and then stimulated for 1, 4, 10 or 24 hours with PDGF-BB (10 ng/ml in starvation medium), or left untreated for the same periods of time. Total RNA was isolated using the RNeasy kit (Qiagen). The RNA quality was checked by formaldehyde agarose gel electrophoresis (RNeasy protocol, Qiagen). Total RNA (40 mg) from cells treated for a given period of time with PDGF or starvation medium alone were labelled in reverse transcription reactions (Superscript II kit, Invitrogen) with dCTP-Cy5 and dCTP-Cy3, respectively (Amersham). In every second replicate experiment the fluorescent deoxynucleotides were swapped. Purified cDNA probes labelled with Cy3 and Cy5 were mixed per pair, and hybridized to cDNA microarray chips (hver1.2.1) from the Sanger Institute/LICR/CRUK Consortium (see http://www.sanger.ac.uk/Projects/Microarrays/ for details and hybridization protocols). For each time point, we performed at least four independent hybridizations, and used at least two different batches of RNA. Chips were scanned in a Perkin Elmer/GSI Lumonics ScanArray 4000 scanner and spot intensities were measured using the QuantArray software (histogram method with background subtraction). Normalization and statistical analysis of the quadruplicate data sets were performed using GeneSpring 5.0 analysis software (Silicon Genetics). A Lowess non-linear normalization was applied and the median of the ratio distribution for each array was set to 1. Regulated spots were selected based on the average ratio values above 1.750 for up-regulated genes and below 0.571 for down-regulated genes. In addition, we considered only genes that were significantly regulated (t-test, p<0.05) based on replicate hybridizations (global error model, GeneSpring). For all features selected using this protocol, the signal was significantly above the background, indicating that the expression of these genes was detectable. Finally, Hver1.2.1 microarrays contain replicate spots (1 to 6) corresponding to the same gene. Genes represented by spots that were not regulated in a similar manner were discarded. We show the average ratio of one representative spot for each regulated gene, with standard error calculated from multiple hybridizations and with the annotation provided by the microarray facility (Hver1.2.1_NCBI33).

Project description:Although immune mechanisms have been described for Peyer’s patch function, in discriminating food nutrients and commensal microflora from pathogenic challenge, an understanding of gene expression patterns associated with normal intestinal homeostasis is lacking. The effects of commensal bacterial colonization on global gene expression in the small intestine of germ-free mice have been described (1, 2). However, no studies have compared expression patterns of various healthy lymphoid tissues in conventionally-raised pigs or other species. The objective of this study was to investigate the pattern of normal gene expression in jejunal Peyer’s patches (JPP) of healthy pigs. We hypothesized that gene expression of intact jejunal Peyer's patch is characterized by genes associated with absorption and secretory functions as well as genes associated with GALT-specific immune functions. Eight hybridizations were performed, with dye swaps of JPP mRNA from four juvenile pigs. Each hybridization compared JPP from an individual pig to the jejunal mesenteric lymph node reference sample, which was pooled from all juvenile pigs in the study. However, two of the hybridizations resulted in poor quality data, and were excluded from further analysis. Normalization, hierarchical clustering, data visualization, and statistical analysis were performed by GeneSpring version 6.2 (Silicon Genetics, Redwood, CA). GeneSpring standardized the intensities for each spot by subtracting the local background, and then normalized globally by locally weighted linear regression (Lowess). A Lowess curve was fit to the log-intensity versus log-ratio plot. 20.0% of the data was used to calculate the Lowess fit at each point. This curve was used to adjust the control value for each measurement. If the control channel was lower than 10 then 10 was used instead. Spots with a flag value of “0” were excluded. The intensity of negative control DNA-containing spots was used to additionally exclude low intensity data. Negative controls include the gene for Arabidopsis thaliana homeodomain-like protein (AY054571); 200 ng/microl Poly(dA) (Amersham, Piscataway, NJ); a 461 bp fragment of the kanamycin-resistant gene, amplified from the pET24b+ plasmid (Novagen, Madison, WI); PRRS virus strain VR-2332 (PRU87392) ORFs 2, 3, 4, 5, 6 and 7; pCMVSport 6 plasmid (Invitrogen, Carlsbad, CA); and pGEM-T plasmid (Promega, Madison WI). The mean intensity of the negative controls for both Cy3 and Cy5 was calculated for each slide, and spots with a mean intensity less than the mean intensity + 2SD were excluded from further analysis. After normalization, GeneSpring averaged replicate spots for each clone across hybridizations. GeneSpring used the Cross Gene Error Model, which was based on replicate values. A Student’s t-test with the Benjamini and Hochberg false discovery rate multiple testing correction (MTC) verified the difference between the natural log of the normalized gene expression ratio (JPP:j-MLN) and a ratio of 1.0. p-values less than 0.05 were considered significant. Series_references: 1.Fukushima K, Ogawa H, Takahashi K, Naito H, Funayama Y, Kitayama T, Yonezawa H, and Sasaki I. Non-pathogenic bacteria modulate colonic epithelial gene expression in germ-free mice. Scand J Gastroenterol 38: 626-634, 2003. 2. Hooper LV, Wong MH, Thelin A, Hansson L, Falk PG, and Gordon JI. Molecular analysis of commensal host-microbial relationships in the intestine. Science 291: 881-884, 2001. Keywords: other

Project description:Although immune mechanisms have been described for Peyer’s patch function, in discriminating food nutrients and commensal microflora from pathogenic challenge, an understanding of gene expression patterns associated with normal intestinal homeostasis is lacking. The effects of commensal bacterial colonization on global gene expression in the small intestine of germ-free mice have been described (1, 2). However, no studies have compared expression patterns of various healthy lymphoid tissues in conventionally-raised pigs or other species. The objective of this study was to investigate the pattern of normal gene expression in jejunal Peyer’s patches (JPP) of healthy pigs. We hypothesized that gene expression of intact jejunal Peyer's patch is characterized by genes associated with absorption and secretory functions as well as genes associated with GALT-specific immune functions. Eight hybridizations were performed, with dye swaps of JPP mRNA from four juvenile pigs. Each hybridization compared JPP from an individual pig to the jejunal mesenteric lymph node reference sample, which was pooled from all juvenile pigs in the study. However, two of the hybridizations resulted in poor quality data, and were excluded from further analysis. Normalization, hierarchical clustering, data visualization, and statistical analysis were performed by GeneSpring version 6.2 (Silicon Genetics, Redwood, CA). GeneSpring standardized the intensities for each spot by subtracting the local background, and then normalized globally by locally weighted linear regression (Lowess). A Lowess curve was fit to the log-intensity versus log-ratio plot. 20.0% of the data was used to calculate the Lowess fit at each point. This curve was used to adjust the control value for each measurement. If the control channel was lower than 10 then 10 was used instead. Spots with a flag value of “0” were excluded. The intensity of negative control DNA-containing spots was used to additionally exclude low intensity data. Negative controls include the gene for Arabidopsis thaliana homeodomain-like protein (AY054571); 200 ng/microl Poly(dA) (Amersham, Piscataway, NJ); a 461 bp fragment of the kanamycin-resistant gene, amplified from the pET24b+ plasmid (Novagen, Madison, WI); PRRS virus strain VR-2332 (PRU87392) ORFs 2, 3, 4, 5, 6 and 7; pCMVSport 6 plasmid (Invitrogen, Carlsbad, CA); and pGEM-T plasmid (Promega, Madison WI). The mean intensity of the negative controls for both Cy3 and Cy5 was calculated for each slide, and spots with a mean intensity less than the mean intensity + 2SD were excluded from further analysis. After normalization, GeneSpring averaged replicate spots for each clone across hybridizations. GeneSpring used the Cross Gene Error Model, which was based on replicate values. A Student’s t-test with the Benjamini and Hochberg false discovery rate multiple testing correction (MTC) verified the difference between the natural log of the normalized gene expression ratio (JPP:j-MLN) and a ratio of 1.0. p-values less than 0.05 were considered significant. Series_references: 1.Fukushima K, Ogawa H, Takahashi K, Naito H, Funayama Y, Kitayama T, Yonezawa H, and Sasaki I. Non-pathogenic bacteria modulate colonic epithelial gene expression in germ-free mice. Scand J Gastroenterol 38: 626-634, 2003. 2. Hooper LV, Wong MH, Thelin A, Hansson L, Falk PG, and Gordon JI. Molecular analysis of commensal host-microbial relationships in the intestine. Science 291: 881-884, 2001.

Project description:Data analysis. Spot detection and matching were performed with a comparative cross analysis of all the gels using DeCyder software v.6.5 (GE Healthcare). 178 spots were selected based on 1.15-fold for protein ratio cut-off, allowing for the appearance of the spots in 23 out of 28 gels (69 out of 84 total images). Data from 95 spots were submitted. 93 spots were identified with high confidence. Spot picking and Trypsin digestion. The spots of interest were picked up by Ettan Spot Picker (GE Healthcare) based on the in-gel analysis and spot picking design by DeCyder software. The gel spots were washed a few times then digested in-gel with modified porcine trypsin protease (Promega, Fitchburg, WI). The digested tryptic peptides were desalted using a Zip-tip C18 (Millipore, Billerica, MA). Peptides were eluted from the Zip-tip with 0.5 uL of matrix solution (alpha-cyano-4-hydroxycinnamic acid, 5 mg/mL in 50% acetonitrile, 0.1% trifluoroacetic acid, 25mM ammonium bicarbonate) and spotted on a MALDI plate. Mass Spectrometry. MALDI-TOF MS and TOF/TOF tandem MS/MS were performed on AB SCIEX TOF/TOF 5800 System (AB SCIEX). MALDI-TOF mass spectra were acquired in reflectron positive ion mode, averaging 4000 laser shots per spectrum. TOF/TOF tandem MS fragmentation spectra were acquired for each sample, averaging 4000 laser shots per fragmentation spectrum on each of the 7-10 most abundant ions present in each sample (excluding trypsin autolytic peptides and other known background ions). Database search. Both the resulting peptide mass and the associated fragmentation spectra were submitted to GPS Explorer workstation equipped with MASCOT search engine (Matrix Science, Boston, MA) to search the Swiss-Prot database. Searches were performed without constraining protein molecular weight or isoelectric point, with variable carbamidomethylation of cysteine and oxidation of methionine residues, and with one missed cleavage also allowed in the search parameters. Candidates with either protein score C.I.% or Ion C.I.% greater than 95 were considered significant. When multiple IDs were significant for a given spot, the selection was made by evaluating apparent molecular weight, isoelectric point, the location of the spot in the gel, and the presence of strips of multiple protein isoforms in the adjacent spots.

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