RNA expression at chromosome 8q24 in human colon
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Mark Pomerantz
PROVIDER: E-GEOD-16228 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Molecular and genomic analysis of microscopic quantities of tumor from formalin-fixed and paraffin-embedded (FFPE) biopsies has many unique challenges. Here we evaluated the feasibility of obtaining transcriptome-wide RNA expression to measure prognostic classifiers from diagnostic prostate needle core biopsies. 158 samples from diagnostic needle core biopsies (Bx) and radical prostatectomies (RP) were collected from 33 patients at three hospitals, each patient provided up to 6 tumor and benign samples. Genome-wide transcriptomic profiles were generated using Affymetrix Human Exon arrays for comparison of gene expression alterations and prognostic signatures between the Bx and RP samples. For 23 patients from UCSF and CSMC, six prostate tissue samples were obtained from each patient: tumor biopsy, tumor RP, benign adjacent biopsy, benign adjacent RP, and benign contralateral biopsy, and benign contralateral RP. For the 10 UHN patients only tumor biopsy and tumor RP samples were obtained. A total of 147 samples passed RNA, cDNA, and microarray quality control.
2016-03-09 | E-GEOD-72220 | biostudies-arrayexpress
Project description:In the study of gene expression regulation in plants, it is often desirable to distinguish transcript pools derived from different alleles present in the same organism. We report here an oligonucleotide tiling microarray designed to specifically target 518 single nucleotide polymorphisms (SNPs) between the two sequenced rice (Oryza sativa) subspecies indica and japonica. The tiling array includes all pairs of 25-mer allele-specific probes interrogating each SNP by placing the polymorphic site at all 25 possible positions within the probe. Hybridization of the tiling array to a titration series in which the japonica- and indica-derived cDNA templates are mixed with altering proportions and development of a novel bioinformatic methodology allowed us to screen for diagnostic probe pairs for each SNP. Our result indicates that 284 (55%) SNPs have at least one diagnostic probe pair suitable for distinguishing and quantifying the relative abundance of allele-specific transcripts. As a proof-of-concept, we analyzed allele-specific expression in reciprocal indica × japonica F1 hybrids and detected imbalanced expression at 95 (33%) and 71 (25%) SNPs, respectively. Comparison results from RNA-sequencing and allele-specific real-time PCR experiment validates the sensitivity and reliability of the tiling array method. Together, our results demonstrate the advantages of the tiling array method in interrogating large numbers of SNPs and for the reliability of the experimental and analytical techniques used for quantifying allele-specific gene expression. For each selected SNP, 25 pairs of 25-mer oligonucleotide probes were designed with one match perfectly to the indica allele and the other the japonica allele within each pair. Among the 25 probe pairs, the polymorphic site was placed at a different position such that all possible 25-mer allele-specific probes spanning the polymorphic site were included. The resultant 25,900 probes were synthesized in triplicate in a single microarray produced on the Maskless Array Synthesizer platform as previously described (Li et al., 2005; 2006). Poly(A+) RNA from japonica, indica and their reciprocal hybrids was reverse transcribed using an oligo(dT)18 primer, during which amino-allyl-modified dUTP (aa-dUTP) was incorporated. The aa-dUTP decorated cDNA was fluorescent labeled by conjugating the monofunctional Cy3 dye (GE Healthcare) to the amino-allyl functional groups in the cDNA. Dye-labeled target was quantified using a spectrophotometer and two μg used for hybridization to each array. In the titration series experiment, the dye-labeled targets from japonica and indica were mixed in known proportions before hybridization.
2010-07-01 | E-GEOD-20678 | biostudies-arrayexpress
Project description:We report here a transcriptonal analysis in six different organ types of a approximately 1 Mb region in soybean (Glycine max) which is sytenic with legume (Medicago truncatula). We used oligonucleotide tiling microarrays to detecte transcription of over 80% of the predicted genes in both species. We detected differential gene expression in the six examined organ types. Keywords: RNA Both the barrel medic and soybean tiling arrays were produced on the Maskless Array Synthesizer platform. Briefly, tiling-paths consisting of 36-mer oligonucleotides offset by five nucleotides were designed to represent both DNA strands of the selected barrel medic and soybean genome sequence. Probes were synthesized at a feature-density of 390,000 probes per array in a âchess boardâ design. Microarray production and storage were carried out. Total RNA and mRNA were sequentially isolated using the RNeasy Plant Mini kit (Qiagen, Valencia, CA) and the Oligotex mRNA kit (Qiagen) according to the manufacturerâs recommendations, respectively. mRNA from different organ types was reverse transcribed using a mixture of oligo(dT)18 and random nonamer primers, during which amino-allyl-modified dUTP (aa-dUTP) was incoporated. The aa-dUTP decorated cDNA was fluorescent labeled by conjugating the monofunctional Cy3 dye (GE Healthcare, Piscataway, NJ) to the amino-allyl functional groups in the cDNA. Two μg dye-labeled targets were used for hybridization.
2010-05-26 | E-GEOD-10056 | biostudies-arrayexpress
Project description:This SuperSeries is composed of the following subset Series: GSE10055: Transcriptional Analysis of 1M Regions in Medicago truncatula Using Tiling Microarrays GSE10056: Transcriptional Analysis of 1M Regions in Glycine max Using Tiling Microarrays Keywords: tiling array, transcriptional analysis Both the barrel medic and soybean tiling arrays were produced on the Maskless Array Synthesizer platform. Briefly, tiling-paths consisting of 36-mer oligonucleotides offset by five nucleotides were designed to represent both DNA strands of the selected barrel medic and soybean genome sequence. Probes were synthesized at a feature-density of 390,000 probes per array in a âchess boardâ design. Microarray production and storage were carried out. Total RNA and mRNA were sequentially isolated using the RNeasy Plant Mini kit (Qiagen, Valencia, CA) and the Oligotex mRNA kit (Qiagen) according to the manufacturerâs recommendations, respectively. mRNA from different organ types was reverse transcribed using a mixture of oligo(dT)18 and random nonamer primers, during which amino-allyl-modified dUTP (aa-dUTP) was incoporated. The aa-dUTP decorated cDNA was fluorescent labeled by conjugating the monofunctional Cy3 dye (GE Healthcare, Piscataway, NJ) to the amino-allyl functional groups in the cDNA. Two μg dye-labeled targets were used for hybridization.
2010-05-26 | E-GEOD-10151 | biostudies-arrayexpress
Project description:We report here a transcriptonal analysis in six different organ types of a approximately 1 Mb region in legume plants barrel medic (Medicago truncatula) which is sytenic with soybean (Glycine max). We used oligonucleotide tiling microarrays to detecte transcription of over 80% of the predicted genes in both species. We detected differential gene expression in the six examined organ types. Keywords: RNA Both the barrel medic and soybean tiling arrays were produced on the Maskless Array Synthesizer platform. Briefly, tiling-paths consisting of 36-mer oligonucleotides offset by five nucleotides were designed to represent both DNA strands of the selected barrel medic and soybean genome sequence. Probes were synthesized at a feature-density of 390,000 probes per array in a âchess boardâ design. Microarray production and storage were carried out. Total RNA and mRNA were sequentially isolated using the RNeasy Plant Mini kit (Qiagen, Valencia, CA) and the Oligotex mRNA kit (Qiagen) according to the manufacturerâs recommendations, respectively. mRNA from different organ types was reverse transcribed using a mixture of oligo(dT)18 and random nonamer primers, during which amino-allyl-modified dUTP (aa-dUTP) was incoporated. The aa-dUTP decorated cDNA was fluorescent labeled by conjugating the monofunctional Cy3 dye (GE Healthcare, Piscataway, NJ) to the amino-allyl functional groups in the cDNA. Two μg dye-labeled targets were used for hybridization.
2010-05-26 | E-GEOD-10055 | biostudies-arrayexpress
Project description:The 1 Mb tiling array was used to represent the 10.0 Mb to 11.0 Mb region of japonica rice Chromosome 10. A tile path was designed with 36-mer probes at a step of 5 nt. All 388,994 probes were retained and synthesized into a single array. This array was hybridized to the cDNA target used for the full-genome tiling array as well as targets prepared from non-coding RNA sample I and II. Keywords: noncoding RNA Non-coding RNA species were isolated from total RNA as previously described (5). Briefly, total RNA isolated from japonica rice leaves using the Trizol method was loaded onto an Qiagen-tip and eluted with 0.6~1.0M NaCl gradient of QRW2 buffer (Qiagen) at 50 oC. Fractions corresponding to tRNAs (sample I) and small RNAs of 80-500 nucleotides (nt) (sample II) were collected. To remove remaining mRNAs and rRNAs, the small RNA fractions were hybridized to a mixture of specifically designed oligos in the binding buffer. Unwanted RNA molecules were targeted and removed by a magnetic bead based process (MicrobExpress kit, Ambion). The enriched non-coding RNA pool was then dephosphorylated with a calf intestine alkaline phosphatase and ligated to a 3ï¢ adaptor oligonucleotide by the T4 RNA ligase (5). Small size molecules and excessive adaptors were removed from the ligation products using the mirVana miRNA isolation kit (Ambion) and reverse transcribed at 50 oC using the Thermoscript (Invitrogen) and the oligo 3RT as the primer (5). Amino-allyl-modified dCTP was incorporated into the cDNA strand during reverse transcription.
2010-06-11 | E-GEOD-6922 | biostudies-arrayexpress
Project description:Multiple discrete regions at 8q24 were recently shown to contain alleles that predispose to many cancers including prostate, breast, and colon. These regions are far from any annotated gene and their biological activities have been unknown. Here we profiled a 5-megabase chromatin segment encompassing all the risk regions for RNA expression, histone modifications, and locations occupied by RNA polymerase II and androgen receptor (AR). This led to the identification of several transcriptional enhancers, which were verified using reporter assays. Two enhancers in one risk region were occupied by AR and responded to androgen treatment; one contained a single nucleotide polymorphism (rs11986220) that resides within a FoxA1 binding site, with the prostate cancer risk allele facilitating both stronger FoxA1 binding and stronger androgen responsiveness. The study reported here exemplifies an approach that may be applied to any risk-associated allele in non-protein coding regions as it emerges from genome-wide association studies to better understand the genetic predisposition of complex diseases. 20 Normal Prostate cDNA samples (no replicates), 4 cell line cDNA samples (each cell line in duplicate), 4 cell line Acetylated H3 ChIP (each line in duplicate), 2 cell line various histone modification and protein ChIPs (6 Abs LNCaP, 5 Abs PC3, each in duplicate). All samples were hybridized with matching genomic DNA as reference.
2009-12-18 | E-GEOD-19561 | biostudies-arrayexpress
Project description:Background The dramatic increase in incidence of ductal carcinoma in situ (DCIS) associated with mammographic screening for breast cancer has given emphasis to the challenges of managing this important clinical entity. Unlike invasive breast cancer, there is no established histopathologic grading system for DCIS, nor are there biological markers of prognosis to guide clinical management. The aim of this study is to use molecular profiling to identify robust and clinically applicable indicators of DCIS malignant potential. Methods Areas of intraduct carcinoma, atypical ductal hyperplasia and benign epithelium were isolated from 46 well-characterised invasive breast cancers by laser capture microdissection. Microarray based gene expression profiling was used to identify genes differentially expressed between DCIS associated with grade 1 and grade 3 invasive carcinoma (‘grade associated genes’). The expression profile of these genes was then determined in all samples and used to define ‘molecular grade’ categories. The genomic basis of gene expression derived categories was examined by array-based comparative genomic hybridisation (CGH). Results DCIS samples could be divided into two subgroups, designated low and high molecular grade (MG) on the basis of expression at 173 grade associated oligonucleotide probes. The low MG subgroup included 21 DCIS samples of low (n=10) and intermediate (n=11) nuclear grade as well as all samples of ADH (n=4) and benign epithelium (n=7). The high MG subgroup included 27 DCIS samples of intermediate (n=7) and high (n=19) nuclear grade. Array CGH revealed distinct differences in the character and degree of genomic aberration associated with MG and the clinical significance of MG was verified by a strong correlation with survival in two independent invasive breast cancer gene expression datasets (n=295 and n=186). MG categories were strongly associated with histopathologic and biomarker features of DCIS. Using a classification tree model, DCIS MG could be accurately predicted in 44/46 (95.7%) of samples using a combination of nuclear grade and Ki67 score. Conclusions Molecular profiling indicates a binary grading scheme for DCIS that is both biologically relevant and clinically informative. The low and high MG DCIS classification could be recapitulated by a novel combination of routinely accessible features. This practical approach has potential to immediately improve clinical evaluation and management of DCIS. Keywords: Paired gene expression and CGH Paired CGH and Gene Expression on DCIS of the breast
2008-11-24 | E-GEOD-7882 | biostudies-arrayexpress
Project description:The objective of the study is to obtain a high resolution transcription map of S. pneumoniae genome and make it available through a genome browser. The study also help to identify gene expression pattern, presence of small RNAs, Antisense expression and operon structure. S. pneumoniae strain TIGR4 (ref) was grown in THYmedium, Todd-Hewitt broth supplemented with 0.5% yeast extract. Cells were harvested from duplicate cultures from mid-log phase (OD600 nm, 0.4â0.6) of growth by centrifugation. The harvested pellets were washed twice in sterile PBS (pH 7.0) and stored at -80°C. RNA was purified from frozen bacterial pellets using Qiagen RNeasy kit by following the manufacturerâs protocol. Isolated RNA was treated with DNase to remove genomic DNA contamination, and purity was checked by performing a one-step RT-PCR using primers specific for 16S rRNA in presence or absence of reverse transcriptase. RNA concentration and quality were determined by using Agilent Bioanalyzer. Purified RNA was stored in nuclease free water at -80°C. One microgram of total RNA was used by Nimblegen systems for labeling and hybridization. 2 biological replicates (single colored) were used in the analysis. 20,000 random probes were designed on the chip as a negative controls to measure non-specific hybridization. 35 genes identified through proteomics of same sample were used as positive controls.
2010-05-06 | E-GEOD-12636 | biostudies-arrayexpress
Project description:Prostate cancer-associated stroma (CAS) plays an active role in malignant transformation, tumor progression, and metastasis. Molecular analyses of CAS have demonstrated significant changes in gene expression; however, conflicting evidence exists on whether genomic alterations in benign cells comprising the tumor microenvironment (TME) underlie gene expression changes and oncogenic phenotypes. This study evaluates the nuclear and mitochondrial DNA integrity of prostate carcinoma cells, CAS, matched benign epithelium and benign epithelium-associated stroma by whole genome copy number analyses, targeted sequencing of TP53, and fluorescence in situ hybridization. Comparative genomic hybridization (aCGH) of CAS revealed a copy-neutral diploid genome with only rare and small somatic copy number aberrations (SCNAs). In contrast, several expected recurrent SCNAs were evident in the adjacent prostate carcinoma cells, including gains at 3q, 7p, and 8q, and losses at 8p and 10q. No somatic TP53 mutations were observed in CAS. Mitochondrial DNA (mtDNA) extracted from carcinoma cells and stroma identified 23 somatic mtDNA mutations in neoplastic epithelial cells but only one mutation in stroma. Finally, genomic analyses identified no SCNAs, no loss of heterozygosity (LOH) or copy-neutral LOH in cultured cancer-associated fibroblasts (CAFs), which are known to promote prostate cancer progression in vivo. Agilent whole human genome aCGH oligonucleotide microarrays were used to assess copy number aberrations in 79 specimens from 20 patients as well as two pairs of cancer-associated fibroblasts (CAF) and benign/normal associated fibroblasts (NAF). 3 CAF/NAF sample pairs were also assessed for DNA copy number aberrations and copy-neutral LOH using the Infinium HumanOmniExpressExome BeadChip Kit.
2016-02-12 | E-GEOD-76456 | biostudies-arrayexpress