Unknown,Transcriptomics,Genomics,Proteomics

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Pharmacogenomics of IFNb therapy

ABSTRACT: This experiment set is used for the manuscript entitled: Pharmacogenomics of Interferon-b therapy in multiple sclerosis: Baseline IFN signature determines pharmacological differences between patients. In this study we generated and analyzed pre- and post- IFNb treatment gene expression patterns of RRMS patients with the aim of identifying pre-existing and/or drug-induced signatures that will allow us to make predictions on the expected pharmacological effects of IFNb treatment. We show that the expression level of IFN response genes prior to treatment, determines the pharmacological differences between patients with MS at the molecular level. A group of 16 Dutch patients (10 females and 6 males) with clinically definite relapsing-remitting MS was recruited from the outpatient clinic of the MS Centre Amsterdam. Mean age at start of IFNb therapy is 40.6 +/- 7.7, mean EDSS is 2.3 +/- 1.3 (range 1-6). Blood samples were obtained just before treatment and 1 month after start of the therapy. Patients received Avonex, Betaferon, Rebif 22 or Rebif 44. A compound treatment design type is where the response to administration of a compound or chemical (including biological compounds such as hormones) is assayed. Compound Based Treatment: Before and 1 month after IFNb therapy Keywords: compound_treatment_design Complex

ORGANISM(S): Homo sapiens

SUBMITTER: Cornelis Verweij

PROVIDER: E-GEOD-10655 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Pharmacogenomics of interferon-beta therapy in multiple sclerosis: baseline IFN signature determines pharmacological differences between patients.

van Baarsen Lisa G M LG Vosslamber Saskia S Tijssen Marianne M Baggen Josefien M C JM van der Voort Laura F LF Killestein Joep J van der Pouw Kraan Tineke C T M TC Polman Chris H CH Verweij Cornelis L CL

PloS one 20080402 4

<h4>Background</h4>Multiple sclerosis (MS) is a heterogeneous disease. In order to understand the partial responsiveness to IFNbeta in Relapsing Remitting MS (RRMS) we studied the pharmacological effects of IFNbeta therapy.<h4>Methodology</h4>Large scale gene expression profiling was performed on peripheral blood of 16 RRMS patients at baseline and one month after the start of IFNbeta therapy. Differential gene expression was analyzed by Significance Analysis of Microarrays. Subsequent expressio ...[more]

PMID: 18382694

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Project description:exceptional example of evolutionary innovation is the single-celled seed trichome in Gossypium ("cotton fiber"). We have used fiber development in Gossypium as a system to understand how morphology can rapidly evolve. Fiber has undergone considerable morphological changes between the short, tightly adherent fibers of G. longicalyx and the derived long, spinable fibers of its closest relative, G. herbaceum, that facilitated cotton domestication. We conducted comparative gene expression profiling across a developmental time-course of fibers from G. longicalyx and G. herbaceum using microarrays with ~ 22,000 genes. Expression changes between stages were temporally protracted in G. herbaceum relative to G. longicalyx, reflecting a prolongation of the ancestral developmental program. Gene expression and GO analyses showed that many genes involved with stress responses were up-regulated early in G. longicalyx fiber development. Several candidate genes up-regulated in G. herbaceum have been implicated in regulating redox levels and cell elongation processes. Three genes previously shown to modulate hydrogen peroxide levels were consistently expressed in domesticated and wild cotton species with long fibers but expression was not detected by qRT-PCR in wild species with short fibers. Hydrogen peroxide is important for cell elongation, but at high concentrations it becomes toxic, activating stress processes that may lead to early onset of secondary cell wall synthesis and the end of cell elongation. These observations suggest that the evolution of long spinable fibers in cotton was accompanied by novel expression of genes assisting in the regulation of reactive oxygen species levels. Our data suggest a model for the evolutionary origin of a novel morphology through differential gene regulation causing prolongation of an ancestral developmental program. Keywords: Cotton, fiber, evolution, time-point, comparative genomic hybridization, stress response genes, H2O2 A balanced developmental loop design for microarray analysis was performed. For G. herbaceum (A1) and G. longicalyx (F), four fiber developmental time-points, 5, 10, 20 and 25 DPA were sampled. Within each species, hybridizations were performed between each pair of consecutive developmental stages by labeling one with Cy5 and the other with Cy3, and by closing the loop with a comparison of 25 and 5 dpa. In addition, 2 hybridizations were done between species at each time-point, using a dye swap for each pair. With three biological replications and 16 slides each, we generated gene expression data from a total of 48 microarrays.

Project description:To identify mRNAs associated with human PUM2 protein, we used a modified Ribonucleoprotein-ImmunoPrecipitation Microarray (RIP-Chip) approach on HeLa S3 cancer cells that express PUM2. PUM ribonucleoprotein (RNP) complexes were captured from cell-free extracts with specific anti PUM2 antibody coupled to protein A sepharose beads, and then eluted with SDS-EDTA. To control for non-specifically enriched RNAs, the same procedure was performed with beads that were not coupled with immunoprecipitating antibodies (mock samples). Total RNA was isolated from cell extracts and immunopurified samples with the mirVanaTM PARISTM kit (Ambion). RNA was quantified with a NanoDrop device (Witeg AG). Poly-adenylated RNAs were amplified in the presence of aminoallyl-UTP with Amino Allyl MessageAmp II aRNA kit (Ambion). For this purpose, 500 ng total RNA from extracts and half (50-100 ng) of the immunopurified RNAs were used for amplification. 8 ug of the amplified RNAs (aaRNA) were fluorescently labeled with NHS-monoester Cy3 and Cy5 dyes (GE HealthSciences), except for mock RNA samples, where an aaRNA amount proportional to the yield obtained from corresponding PUM affinity isolates was used.For PUM2 RIPs, we performed four biological replicates but omitted the dye swaps due to the lower aaRNA obtained after amplification (~10 ug aaRNA from PUM2 RIPs, 9 ug aaRNA from mock RIPs). The Cy3- and Cy5-labeled aaRNA samples were mixed and hybridized to human cDNA microarrays. Detailed methods for microarray experiments are available at http://cmgm.stanford.edu/pbrown/protocols/index.html. cDNA microarrays were produced by the Stanford Functional Genomic Facility and contained 43,197 human probes representing 26,524 Unigene cluster IDs (12,466 ENSEMBL annotated genes) spotted on Corning Ultra GAPS slides. Spotted cDNAs were cross-linked with 65 mJ of UV irradiation on slides, which were then post-processed for 1 hour at 42°C in pre-hybridization solution (5x SSC, 0.1% SDS, 0.1 mg/ml BSA), washed twice in 400 ml of 0.1x SSC for 5 min, dunked in 400 ml ultrapure water for 30 sec, and dried by centrifugation at 550 rpm for 5 min. Slides were used the same day. Cy3- and Cy5-labeled aaRNA probes were mixed and applied to arrays in hybridization solution (3x SSC, 20 ug poly(A) RNA [Invitrogen], 20 ug yeast tRNA [Invitrogen], 20 ug Human Cot-1 DNA [Invitrogen], 20 mM HEPES [pH 7.0] and 0.3% SDS) for 18 h at 65°C. The arrays were then washed sequentially in 400 ml of 2x SSC with 0.1% SDS, 1x SSC, and 0.2x SSC. The first wash was performed for 5 min at 65°C, the subsequent washes were performed for 5 min at RT. The arrays were dried by centrifugation and immediately scanned with an AxonScanner 4200A (Molecular Devices). Data were collected using GENEPIX 5.1 (Molecular Devices). Arrays were normalized computationally by the Stanford Microarray Database (SMD). The data were filtered for signal over background of greater than 1.5 in the channel measuring aaRNA from extract, and only features that met these criteria in > 50% of the arrays were included for further analysis. Log2 median ratios were retrieved and exported into Microsoft Excel. To identify transcripts that were specifically enriched by association with PUM2, we performed two class Significance Analysis of Microarrays (SAM) on median centered arrays. A replicate experimental design type is where a series of replicates are performed to evaluate reproducibility or as a pilot study to determine the appropriate number of replicates for a subsequent experiments. Antibody used in IP: 50 ug rabbit anti-PUMILIO 2 (Bethyl Laboratories, #300-202A) Keywords: replicate_design Computed

Dataset Information

Pharmacogenomics of IFNb therapy

Publications

Pharmacogenomics of interferon-beta therapy in multiple sclerosis: baseline IFN signature determines pharmacological differences between patients.

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