Unknown,Transcriptomics,Genomics,Proteomics

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Bovine tissue gene expression profiling at peak lactation

ABSTRACT: Tissue from mammary, liver, and three adipose depots (mesenteric, omental, and subcutaneous) was collected at slaughter from a cow at peak lactation to examine gene expression profiles of 7872 sequences using a bovine cDNA microarray. RNA (20 ug) from tissues and a reference standard derived from a mixture of tissues were used to make aminoallyl-labeled cDNA followed by incorporation of Cy3-ester and Cy5-ester (Amersham, Piscataway, NJ). Each experimental sample was co-hybridized with the reference standard, which allowed the treatment of fluorescence ratios as measurements of relative expression across all samples and time points. Probes were hybridized to the array for 2 days at 42 C. All samples were hybridized to duplicate slides for a total of 4 spots per cDNA element. The microarray platform has already been described [Everts et al. (2005) Vet. Immunol. Immunopathol. 105:235-245]. Slides were scanned for both dye channels with a Scanarray 4000 (GSI-Lumonics, Billerica, MA) dual-laser confocal scanner and images were processed using GenePix 4.0 (Axon Instruments, Inc., CA).

ORGANISM(S): Bos taurus

SUBMITTER: James Drackley

PROVIDER: E-GEOD-4495 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Despite being one of the first eukaryotic transcriptional regulatory elements identified, the sequence of a native TATA box and its significance remain elusive. Applying criteria associated with TATA boxes we queried several Saccharomyces genomes and arrived at the consensus TATA(A/T)A(A/T)(A/G). Approximately 20% of yeast genes contain a TATA box. Strikingly, TATA box-containing genes are associated with responses to stress, are highly regulated, and preferentially utilize SAGA rather than TFIID when compared to TATA-less promoters. Transcriptional regulation in yeast appears to be mechanistically bipolar, possibly reflecting a need to balance inducible stress-related responses with constitutive housekeeping functions. A strain containing amino terminal HA-tagged TBP and its parental untagged counterpart BY4741 (Resgen) were grown at 23?C in CSM to OD600 = 0.8-1.0. Cultures were shifted to 37?C for 5 minutes (to mimic heat treatments used elsewhere (Huisinga and Pugh, 2004)), then crosslinked at 23?C with 1% formaldehyde. ChIP was performed as described previously (Strahl-Bolsinger et al., 1997) with the some modification. Following cell disruption with glass beads, the chromatin was partially purified by centrifugation (Kurdistani and Grunstein, 2003). HA-TBP was immunopurified with 12CA5 antibody. After elution and reversal of the crosslinks, samples were subjected to non-specific amplification, labeling, and array hybridization as described (Bohlander et al., 1992; Chitikila et al., 2002). The amplification procedure was modified as follows. Following 15 round B amplification cycles, DNA was purified via QIAquick PCR Purification kit. Ten cycles were used in round C. Intergenic microarrays were generated by PCR amplification of entire intergenic regions of all yeast genes, then spotted onto glass slides. Spot intensities were filtered to remove any signal that was less then one standard deviation above local background. Three replicates were performed and their log2 ratios (enriched/input) were averaged. Keywords = Chromatin Immunoprecipitation Keywords = genome-wide binding

Project description:Cows were selected from two groups of 12 cows enrolled in a large experiment to assess the effects of ad libitum or restricted intake of moderate-energy diets during the entire dry period on pre-partum metabolism and post-partum metabolism and performance. A corn silage-based diet (26% of diet dry matter) providing 1.59 Mcal/kg during the far-off dry period (first 5 wk of an 8-wk dry period) or providing 1.61 Mcal/kg during the close-up dry period (last 3 wk of the dry period) was fed for ad libitum or restricted intake. Four multiparous Holstein cows were randomly selected from the ad libitum and restricted intake groups. A 7,872-element cDNA microarray spotted in duplicate on amino silane-coated glass slides (Everts et al. 2005, Vet. Immunol. Immunopathol. 105:235-245) was used for transcript profiling. Annotation was based on similarity searches (January, 2005) using sequential BLASTN and TBLASTX against human and mouse UniGene databases and the human genome. The 7,872-element array represents more than 6,300 unique genes. Gene Ontology (GO) terms were parsed from LocusLink to functionally annotate cDNA sequences. RNA (20 ug) from liver tissue and a reference standard derived from a mixture of tissues not including liver were used to make aminoallyl-labeled cDNA followed by incorporation of Cy3-ester and Cy5-ester (Amersham, Piscataway, NJ). Each experimental sample was co-hybridized with the reference standard, which allowed the treatment of fluorescence ratios as measurements of relative expression across all samples and time points. Probes were hybridized to the array for 2 days at 42 C. All samples were hybridized to duplicate slides for a total of 4 spots per cDNA element. A total of 106 microarrays were run to complete the experiment. Slides were scanned for both dye channels with a Scanarray 4000 (GSI-Lumonics, Billerica, MA) dual-laser confocal scanner and images were processed using GenePix 4.0 (Axon Instruments, Inc., CA).

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Bovine tissue gene expression profiling at peak lactation

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