Gene expression analysis following hypoxia–reoxygenation in rat gastric epithelial cells using a high-density oligonucleotide array

Owing to the difficulty in obtaining mammary gland tissue from lactating animals, it is difficult to test the expression levels of genes in mammary gland. The aim of the current study was to identify if milk fat globule (MFG) in buffalo milk was an alternative to mammary gland (MG) and milk somatic cell (MSC) for gene expression analysis. Six buffalos in late lactation were selected to collect MFG and MSC, and then MG was obtained by surgery. MFG was stained with acridine orange to successfully visualise RNA and several cytoplasmic crescents in MFG. The total RNA in MFG was successfully isolated and the integrity was assessed by agarose gel electrophoresis. We analysed the cellular components in MFG, MG and MSC through testing the expression of cell-specific genes by qRT-PCR. The results showed that adipocyte-specific gene (AdipoQ) and leucocyte-specific genes (CD43, CSF1 and IL1α) in MFG were not detected, whereas epithelial cell marker genes (Keratin 8 and Keratin 18) in MFG were higher than in MSC and lower than in MG, fibroblast marker gene (vimentin) in MFG was significantly lower than in MG and MSC, milk protein genes (LALBA, BLG and CSN2) and milk fat synthesis-related genes (ACC, BTN1A1, FABP3 and FAS) in MFG were higher than in MG and MSC. In conclusion, the total RNA in MFG mainly derives from mammary epithelial cells and can be used to study the functional gene expression of mammary epithelial cells.

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High Resolution Epigenomic Mapping of Myelodysplastic Syndrome Reveals a High Level of Functionally Important Methylation.

Blood ◽

10.1182/blood.v108.11.2637.2637 ◽

2006 ◽

Vol 108 (11) ◽

pp. 2637-2637

Author(s):

Li Zhou ◽

Joanna Opalinska ◽

Davendra Sohal ◽

Simrit Parmar ◽

Perry Pahanish ◽

...

Keyword(s):

Gene Expression ◽

Expression Analysis ◽

Gene Expression Analysis ◽

Cpg Island ◽

High Density ◽

High Rate ◽

Comparative Genomic ◽

Differentially Methylated Genes ◽

High Level ◽

Very High

Abstract The myelodysplastic syndromes (MDS) are collections of heterogeneous hematologic diseases characterized by clonal hematopoietic defects. Even though gene expression studies have tried to differentiate between prognostic subgroups, there are concerns with standardization and validity of these studies. Decitabine and 5-azacytidine have shown activity in MDS, but it is unclear if hypomethylation can explain their efficacy. To address these issues we have developed a novel platform that uses a combination of gene expression analysis, high density array based comparative genomic hybridization (aCGH) and genome wide methylation analysis to perform an integrated high throughput epigenomic analysis of MDS. Using this approach, we conducted a pilot study in 5 patients with MDS. Gene expression analysis was performed using 37K oligo maskless arrays and high density aCGH was performed at 6Kb resolution using the Nimblegen platform. Whole genome methylation was analyzed by a recently described novel method ( Khulan et al, Genome Res. 2006 Aug;16(8)) that uses differential methylation specific digestion by HpaII and MspI followed by pcr amplification, two color labeling and hybridization to quantitatively determine individual promoter CpG island methylation. aCGH revealed a very high number of deletions (range 515–2722, mean 1073±937) and amplifications (range 614–1247, mean 843±254) not visualized by conventional cytogenetic analysis. These were also seen on peripheral blood mononuclear cells and correlated with those seen in the bone marrow. 22 common regions were found to amplified and 15 regions commonly deleted in 80% of the samples and contained genes involved in transcription and signaling. A custom human oligo array was used to determine methylation by calculating HpaII/MspI cut fragment intensity ratio. MDS samples demonstrated a very high level of methylation (range 70–84%). Expression was found to be significantly decreased for the genes that were methylated (p<.0001, T test) demonstrating the functional relevance of this assay. Analysis of common differentially methylated genes (when compared to normal samples) and their validation are ongoing. Micro deletions and amplifications seen on aCGH did not correlate with changes in global expression of the involved genes. Interestingly, large deletions (-7q) and large amplifications (+1p) correlated significantly with corresponding changes in gene expression when compared to the rest of the chromosome. (p<.0001, Anova) Integration of methylation data with aCGH data increased the predictive value of corresponding changes in gene expression. This result demonstrates that loss of heterozygosity coupled with the methylation of the remaining allele may result in pathogenic gene silencing in MDS. The high rate of methylation and DNA copy number alterations demonstrated in MDS in our study suggest that this integrated approach mightl be useful to define prognostic subgroups in future studies and aid in gene discovery.

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