Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Transcription profiling of mouse wild-type and B cell-specific TAK1-deficient B cells to elucidate how TAK1 regulates BCR-mediated proliferative response

ABSTRACT: BCR-induced gene expression profile in wild-type and B cell-specific TAK1-deficient B cells; to elucidate how TAK1 regulates BCR-mediated proliferative response Experiment Overall Design: Purified splenic B cells (CD43 negative) were treated with or without anti-IgM (20 micloG/mL) for 4 h. Total RNA was extracted with an RNeasy kit (Qiagen), double-stranded DNA was synthesized from 10 micloG of total RNA with the SuperScript Choice System (Invitrogen) primed with T7-(dT) 24 primer. These cDNAs were used to prepare biotin-labeled cRNA by an in vitro transcription reaction performed using T7 RNA polymerase in the presence of biotinylated-ribonucleotides, according to the manufacturerâs protocol (Enzo Diagnostics). The cRNA product was purified using an RNeasy kit, fragmented, and hybridized to Affymetrix mouse expression array A430.2 microarray chips, according to the manufacturerâs protocol (Affymetrix). The hybridized chips were stained, washed, and scanned with a GeneArray Scanner (Affymetrix).

ORGANISM(S): Mus musculus

SUBMITTER: Shizuo Akira

PROVIDER: E-GEOD-3065 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Essential function for the kinase TAK1 in innate and adaptive immune responses.

Sato Shintaro S Sanjo Hideki H Takeda Kiyoshi K Ninomiya-Tsuji Jun J Yamamoto Masahiro M Kawai Taro T Matsumoto Kunihiro K Takeuchi Osamu O Akira Shizuo S

Nature immunology 20050925 11

Transforming growth factor-beta-activated kinase 1 (TAK1) has been linked to interleukin 1 receptor and tumor necrosis factor receptor signaling. Here we generated mouse strains with conditional expression of a Map3k7 allele encoding part of TAK1. TAK1-deficient embryonic fibroblasts demonstrated loss of responses to interleukin 1beta and tumor necrosis factor. Studies of mice with B cell-specific TAK1 deficiency showed that TAK1 was indispensable for cellular responses to Toll-like receptor lig ...[more]

PMID: 16186825

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Project description:We have used commercially available oligonucleotide arrays to measure the relative expression levels of >10,000 genes and ESTs. Recent evidence suggested certain advantages of performing such expression experiments on two or more different platforms of oligonucleotide arrays. Here we have used two oligonucleotide platforms, MG U74Av2.0 arrays from Affymetrix and CodeLink UniSet1 from GE Healthcare, to assess whole brain gene expression patterns in naive C57BL/6 and DBA/2J mice. C57BL/6J (n = 5) and DBA/2J (n = 4) male mice, 25-30 g body weight, were obtained from Jackson Laboratories. Animals were group-housed and quickly sacrificed by CO2 exposure according to a protocol approved by the UCHSC IACUC. Brains were rapidly removed and stored at -80oC until use. Whole brain was homogenized in lysis buffer using a Polytron, and total RNA was isolated using the RNeasy Midi Kit (Qiagen) following the protocol supplied by the manufacturer. An additional clean-up of total RNA was carried out using the RNeasy Mini Kit (Qiagen). Using the protocol supplied by the manufacturer, double-stranded cDNA was synthesized from the total RNA (4 ug of total RNA from an individual mouse brain was used for each array) and was used to obtain biotin-labeled cRNA by an in vitro transcription reaction. Biotin-labeled cRNA was recovered using the RNeasy kit. Biotin-labeled cRNA from each mouse was then fragmented and hybridized with two separate (duplicate) CodeLink BioArrays. The average of the gene expression intensity values for each probe set from these technical replicates was used for data analysis. The BioArrays were subsequently stained for 30 min with Cy5-Streptavidin and washed prior to scanning. For MG U74Av2 arrays,double-stranded cDNA was synthesized from 5 ug of total RNA, using the protocol supplied by the manufacturer, and used to obtain biotin-labeled cRNA by an in vitro transcription reaction. Biotin-labeled cRNA was then fragmented and the quality of the fragmented cRNA was assessed using Test2 chips prior to hybridization with the Affymetrix arrays. Hybridization of the cRNA with the Affymetrix arrays was carried out according to the manufacturer's protocol. The arrays were stained with streptavidin-phycoerythrin conjugate and scanned by a GeneArray scanner. Keywords: other

Dataset Information

Transcription profiling of mouse wild-type and B cell-specific TAK1-deficient B cells to elucidate how TAK1 regulates BCR-mediated proliferative response

Publications

Essential function for the kinase TAK1 in innate and adaptive immune responses.

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