Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Gene expression profiles in 132 laser microdissected colorectal cancer tissues

ABSTRACT: Tissues from another series of 132 patients with colorectal cancer were collected by laser micro-dissection with the Leica Laser Microdissection System (Leica Microsystems). Gene expression levels obtained from 132 microdissected colorectal tumors were compared with microdissected normal colon epitheliums. we used the commercially available Human Whole Genome Oligo DNA Microarray Kit (Agilent Technologies). Labeled cRNAs were fragmented and hybridized to an oligonucleotide microarray (Whole Human Genome 4×44K Agilent G4112F). Fluorescence intensities were determined with an Agilent DNA Microarray Scanner and were analyzed using G2567AA Feature Extraction Software Version A.7.5.1 (Agilent Technologies).

ORGANISM(S): Homo sapiens

SUBMITTER: Koshi Mimori

PROVIDER: E-GEOD-21815 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Long noncoding RNA HOTAIR regulates polycomb-dependent chromatin modification and is associated with poor prognosis in colorectal cancers.

Kogo Ryunosuke R Shimamura Teppei T Mimori Koshi K Kawahara Kohichi K Imoto Seiya S Sudo Tomoya T Tanaka Fumiaki F Shibata Kohei K Suzuki Akira A Komune Shizuo S Miyano Satoru S Mori Masaki M

Cancer research 20110823 20

The functional impact of recently discovered long noncoding RNAs (ncRNAs) in human cancer remains to be clarified. One long ncRNA which has attracted attention is the Hox transcript antisense intergenic RNA termed HOTAIR, a long ncRNA expressed from the developmental HOXC locus located on chromosome 12q13.13. In cooperation with Polycomb complex PRC2, the HOTAIR long ncRNA is reported to reprogram chromatin organization and promote breast cancer metastasis. In this study, we examined the status ...[more]

PMID: 21862635

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Project description:The mammalian lens consists of an aged core of quiescent cells enveloped by a layers of mature fully elongated and younger, continuously elongating and transcriptionaly active cells. In the present study, we compared global gene expression profiles in elongating and mature, non-elongating fiber cells. The microarray-based comparison of gene expression profiles was performed using Agilent 22K Mouse Oligo microarrays with two rounds of RNA amplification from samples obtained by laser capture microdissection (LCM) of paraffin-embedded lens sections. Lenses were removed and immediately fixed using 4% paraformaldehide or methanol-based UMFIX reagent (Sakura Finetek USA, Inc.). To map the exact location of both elongating and maturing fibers on the lens slices, lenses were vibratome-sliced (Vibratome 1000, St. Louis, MO) as described previously and the GFP expression pattern was captured by confocal microscopy. For RNA extraction lens tissue was sliced into 5 micron-thick paraffin sections and microdissected using LCM (Leica Microsystems, Bannockburn, IL). The mid-saggital slices were used both for syncytium border measurements and for LCM. Control measurement (data not shown) confirmed that similar rates of shrinkage in paraformaldehyde- and UMFIX-fixed samples did not affect precision of LCM dissection. Fiber cell samples were dissected out of 5 micron-thick slices. In one experiment we typically processed about 40 slices, which was sufficient to collect the minimum of 200 zone-specific cells pooled from P5 littermate lenses. This sample size provided a reliable representation of RNA species in experimental procedure originally designed and tested for just 1-10 cells. Localization of the syncytium border characterized by the abrupt change of GFP labeling pattern was captured by confocal microscopy as described previously. In brief, GFP fluorescence was visualized using LSM510 instrument (Carl Zeiss, NY) equipped with an argon/krypton laser at 488 nm excitation and a 515-565 nm band pass emission filter. Physical parameters of the zones containing young and maturing fibers were measured in fixed lens slices using the morhphometric software provided by Zeiss. Lenses were fixed in 4% paraformaldehyde/PBS and sectioned with a vibratome. Cells from elongating and maturing fiber regions were dissected out using the Leica DMLA laser capture microscope, (Leica Microsystems, Bannockburn, IL). The cut-out pieces containing captured cells were put directly into tubes containing the lysis buffer supplied in the Absolutely RNA® nanoprep kit (Stratagene, La Jolla, CA). Total RNA from the microdissected tissue sections was extracted and purified using the Absolutely RNA nanoprep kit according to the manufacturer's protocol. Caps briefly placed onto the section without laser activation were used as negative controls. Samples from several age-matched lenses were pooled together to obtain differentiation-specific samples for microarray analysis. Target RNA amplification and labeling with Cy-3 or Cy-5 dyes from CyDye Post Labelling Reactive Dye Pack (Amersham, USA) was carried out in two rounds using the Amino Allyl MessageAmp™ aRNA Kit (Ambion, USA) as specified by the manufacturer. Quality and size distribution of the targets were determined by the Agilent 2100 Bioanalyzer (Agilent Technologies, USA). The Amino Allyl MessageAmp aRNA Kit is configured to incorporate the modified nucleotide, 5-(3-aminoallyl)-UTP (aaUTP) into the aRNA during in vitro transcription. Once purified and fragmented, the dye labeled aRNA was used for microarray hybridization. Labeled amplified RNA was hybridized to the 22K Mouse Oligo microarrays (Agilent Technologies)) according to the manufacturer's instructions. After hybridization microarrays were washed and scanned using GenePix 4000A (Axon Instruments,Inc.). Keywords = Lens Keywords = differentiation Keywords = microarray Keywords = gene expression profiling Keywords = laser capture microdissection Keywords = RNA amplification Keywords: repeat sample

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Dataset Information

Gene expression profiles in 132 laser microdissected colorectal cancer tissues

Publications

Long noncoding RNA HOTAIR regulates polycomb-dependent chromatin modification and is associated with poor prognosis in colorectal cancers.

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