Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of mouse adult male wild-type liver (M-WT), adult female wild-type liver (F-WT), adult male liver-specific HNF4alpha knockout liver (M-KO) and adult female liver-specific HNF4alpha knockout liver (F-KO) to investigate sex-dependent and HNF4alpha-dependent gene expression

ABSTRACT: A series of dual-channel gene expression profiles obtained using Rosetta/Agilent Whole Mouse Genome oligonucleotide microarrays, 4 x 44K format, was used to identify sex-dependent and HNF4alpha-dependent differences in gene expression in adult mouse liver. This series is comprised of four sex-genotype combinations: adult male wild-type liver (M-WT), adult female wild-type liver (F-WT), adult male liver-specific HNF4alpha knockout liver (M-KO) and adult female liver-specific HNF4alpha knockout liver (F-KO). Four pools, each comprised of 4 randomly selected individual liver RNAs, were prepared for each sex-genotype combination. The pools were paired randomly to generate 4 separate experimental comparisons: M-WT:F-WT (first array comparison), M-WT:M-KO (second array comparison), F-WT:F-KO (third array comparison), and M-KO:F-KO (fourth array comparison). A total of 4994 HNF4alpha-dependent genes were identified, of which ~1000 fewer genes responded to the loss of HNF4alpha in female liver as compared to male liver. Moreover, 90% of the genes showing sex-specific expression in the liver were shown to lose sex specificity in HNF4alpha-deficient liver. Experiment Overall Design: An Alexa555-labeled cDNA sample is co-hybridized with an Alexa647-labeled cDNA sample. The samples are then dye-swapped and compared again on a second microarray chip. Together, these two mixed cDNA samples are considered a fluorescent reverse pair (dye swap). Similarly, a second fluorescent reverse pair is generated and the two pairs are averaged. The normalized expression ratio for each array is reported along with the two separate intensities. In this way, dye swaps were carried out for each of the four experimental comparisons. Thus, four microarrays, one for each mixed cDNA sample, were hybridized for each of the four fluorescent reverse pairs, giving a total of 16 microarrays.

ORGANISM(S): Mus musculus

SUBMITTER: David Waxman

PROVIDER: E-GEOD-10390 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:A series of two color gene expression profiles obtained using Agilent 44K expression microarrays was used to examine sex-dependent and growth hormone-dependent differences in gene expression in rat liver. This series is comprised of pools of RNA prepared from untreated male and female rat liver, hypophysectomized (‘Hypox’) male and female rat liver, and from livers of Hypox male rats treated with either a single injection of growth hormone and then killed 30, 60, or 90 min later, or from livers of Hypox male rats treated with two growth hormone injections spaced 3 or 4 hr apart and killed 30 min after the second injection. The pools were paired to generate the following 6 direct microarray comparisons: 1) untreated male liver vs. untreated female liver; 2) Hypox male liver vs. untreated male liver; 3) Hypox female liver vs. untreated female liver; 4) Hypox male liver vs. Hypox female liver; 5) Hypox male liver + 1 growth hormone injection vs. Hypox male liver; and 6) Hypox male liver + 2 growth hormone injections vs. Hypox male liver. A comparison of untreated male liver and untreated female liver liver gene expression profiles showed that of the genes that showed significant expression differences in at least one of the 6 data sets, 25% were sex-specific. Moreover, sex specificity was lost for 88% of the male-specific genes and 94% of the female-specific genes following hypophysectomy. 25-31% of the sex-specific genes whose expression is altered by hypophysectomy responded to short-term growth hormone treatment in hypox male liver. 18-19% of the sex-specific genes whose expression decreased following hypophysectomy were up-regulated after either one or two growth hormone injections. Finally, growth hormone suppressed 24-36% of the sex-specific genes whose expression was up-regulated following hypophysectomy, indicating that growth hormone acts via both positive and negative regulatory mechanisms to establish and maintain the sex specificity of liver gene expression. For full details, see V. Wauthier and D.J. Waxman, Molecular Endocrinology (2008) This series is comprised of pools of liver RNA prepared from untreated male, hypophysectomized (‘Hypox’) male, untreated female and Hypox female rats (3-4 livers/pool), as well as liver RNA prepared from Hypox male rats treated with a single growth hormone injection and killed either 30, 60, or 90 minutes later (pool of n = 4 livers) or from Hypox male rats treated with two growth hormone injections spaced 3 or 4 hr apart (pool of n = 5 livers). The pools were paired to generate the following 6 direct microarray comparisons: 1) untreated male liver vs. untreated female liver; 2) Hypox male liver vs. untreated male liver; 3) Hypox female liver vs. untreated female liver; 4) Hypox male liver vs. Hypox female liver; 5) Hypox male liver + 1 growth hormone injection vs. Hypox male liver; and 6) Hypox male liver + 2 growth hormone injections vs. Hypox male liver. Dye swapping experiments were carried out for each of the six hybridization experiments, as follows. The Alexa 555-labeled cDNA from one of the two untreated male pools was mixed with the Alexa 647-labeled cDNA from one of the two untreated female pools. Similarly, Alexa 647-labeled cDNA from the second untreated male pool was mixed with the Alexa 555-labeled cDNA from the second untreated female pool. Together, these two mixed cDNA samples comprise a fluorescent reverse pair (dye swap). Dye swaps were similarly carried out for each of the five other competitive hybridization experiments, except that for experiments 5 and 6, a single pool of M-Hypox + GH liver cDNA, or a single pool of M-Hypox + 2GH liver cDNA, was used in each half of the fluorescent reverse pair. Two microarrays, one for each mixed cDNA sample, were hybridized for each of the six fluorescent reverse pairs, giving a total of 12 microarrays.

Project description:Peumonia is the most common cause of death due to infectious diseases in the western hemisphere. The molecular events associated with pulmonary infections caused by Streptococcus pneumoniae and Influenza A virus are incompletely understood. Pathophysiological and protective processes are initiated by immune receptors specifically recognizing pathogenic structures serving to elicit a qualitatively and quantitatively adequate immune response. To provide a molecular framework towards a better understanding of the processes relevant in severe infectious pneumonia we performed a transcriptome analysis of lungs from mice infected with S. pneumoniae or Influenza A virus. Overall, we detected 1300 genes that exhibit significant differential expression after infection with either pathogen. Of those were approximately 36 % specific for pneumococcal and 30 % specific for the viral infection, yielding pathogen-specific as well as common inflammatory transcriptional signatures. Characteristically, these results resolve not only a differential response on the cytokine and chemokine level, although common induction of type I and type II interferons, TNFa, and IL-6 underlies both infections, but emphasize the potentially important role exerted by many genes implicated in the regulation and fine tuning of the inflammatory response. Furthermore, we noted a specific decrease in B cells after S. pneumoniae infection, which is not solely confined to the lung. Massive induction of apoptosis in pulmonary B cells could reflect a pneumococcal virulence strategy aiming against the lymphocyte population that is of utmost importance for the defence against capsulated bacteria. The pathophysiological consequences of Influenza A virus infection become obvious through differential induction of genes implicated in tissue regeneration and proliferation associated with detection of the emerging protective T cell response. These results may provide new insights into the pathogenesis and protective mechanisms important for the development of improved diagnostic and therapeutic strategies. Microarray analyses were performed as two-colour hybridizations. RNA was labelled with a Fluorescent Linear Amplification Kit (Agilent Technologies). Briefly, 4 Âµg total RNA was reverse transcribed with an oligo-dT-T7 promoter primer and MMLV-RT. Second strand synthesis was carried out with random hexamers. Fluorescent antisense cRNA was synthesized with either cyanine 3-CTP (Cy3-CTP) or cyanine 5-CTP (Cy5-CTP) and T7 polymerase. Purified products were quantified at A552nm for Cy3-CTP and A650nm for Cy5-CTP. Prior hybridization, 1.25 Âµg labelled cRNA each were fragmented, mixed with control targets and hybridization buffer according to the supplier's protocol (Agilent Technologies). Hybridizations were performed for 17 h at 60Â°C. The slides were washed according to the manufacturer's protocol and scanning of the arrays was performed with 5 Âµm resolution using a DNA microarray laser scanner (Agilent Technologies). To compensate for dye specific effects, and to ensure validity of the data, a colour swap analysis (fluorescence reversal) was performed. Features were extracted with the image analysis tool Version A6.1.1.1 from Agilent Technologies. Subsequent data analyses were carried out on the Rosetta Inpharmatics platform Resolver Built 3.2.2. Differential expression patterns were identified by applying 2-fold expression level cut-offs and an anti-correlation of the dye reversal experiments with an error weighted p-value < 0.05.

Dataset Information

Transcription profiling of mouse adult male wild-type liver (M-WT), adult female wild-type liver (F-WT), adult male liver-specific HNF4alpha knockout liver (M-KO) and adult female liver-specific HNF4alpha knockout liver (F-KO) to investigate sex-dependent and HNF4alpha-dependent gene expression

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