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Normal colon epithelium: a dataset for the analysis of gene expression and alternative splicing events in colon disease.

Mojica W, Hawthorn L - BMC Genomics (2010)

Bottom Line: One of the main objectives of this study was to generate a reference gene expression data set for normal colonic epithelium which can be used in comparisons with diseased tissues, as well as to provide a dataset that could be used as a baseline for studies in alternative splicing.For demonstration purposes, we have compared the data derived from these cells to a publically available set of tumor and matched normal colon data.Our analysis of splice variants illustrate that this is a very labor intensive procedure, requiring vigilant examination of the data.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular Oncology Program, Medical College of Georgia Cancer Center, Augusta, GA, USA.

ABSTRACT

Background: Studies using microarray analysis of colorectal cancer have been generally beleaguered by the lack of a normal cell population of the same lineage as the tumor cell. One of the main objectives of this study was to generate a reference gene expression data set for normal colonic epithelium which can be used in comparisons with diseased tissues, as well as to provide a dataset that could be used as a baseline for studies in alternative splicing.

Results: We present a dependable expression reference data set for non-neoplastic colonic epithelial cells. An enriched population of fresh colon epithelial cells were obtained from non-neoplastic, colectomy specimens and analyzed using Affymetrix GeneChip EXON 1.0 ST arrays. For demonstration purposes, we have compared the data derived from these cells to a publically available set of tumor and matched normal colon data. This analysis allowed an assessment of global gene expression alterations and demonstrated that adjacent normal tissues, with a high degree of cellular heterogeneity, are not always representative of normal cells for comparison to tumors which arise from the colon epithelium. We also examined alternative splicing events in tumors compared to normal colon epithelial cells.

Conclusions: The findings from this study represent the first comprehensive expression profile for non-neoplastic colonic epithelial cells reported. Our analysis of splice variants illustrate that this is a very labor intensive procedure, requiring vigilant examination of the data. It is projected that the contribution of this set of data derived from pure colonic epithelial cells will enhance studies in colon-related disease and offer a vital baseline for studies aimed at elucidating the mechanisms of alternative splicing.

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Histology of colonic mucosa. (1a) Section of mucosa was stripped off the colectomy specimen and processed by formalin fixation and paraffin embedding. The mucosa is composed of the lamina propria and epithelium. As is evident, it is not a homogeneous collection of cells, but rather a composite of the cells that are present in the lamina propria (chronic inflammatory cells, smooth muscle cells and vessels) and epithelium (hematoxylin and eosin 10×). (1b) Histologic evidence of enrichment for colonic epithelial cells from the procurement approach described in the text (hematoxylin and eosin 20×).
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Figure 6: Histology of colonic mucosa. (1a) Section of mucosa was stripped off the colectomy specimen and processed by formalin fixation and paraffin embedding. The mucosa is composed of the lamina propria and epithelium. As is evident, it is not a homogeneous collection of cells, but rather a composite of the cells that are present in the lamina propria (chronic inflammatory cells, smooth muscle cells and vessels) and epithelium (hematoxylin and eosin 10×). (1b) Histologic evidence of enrichment for colonic epithelial cells from the procurement approach described in the text (hematoxylin and eosin 20×).

Mentions: To control for variables associated with specimen integrity and sample processing, the time intervals between receipt of the sample in the operating suite and extirpation of each specimen was recorded. Additionally, the time intervals for delivery to the laboratory, placement of exfoliated cells in PBS with DTT, and the endpoint wherein enriched cells were stabilized by snap freezing were also documented and presented in table 3. A portion of the exfoliated cells from one case were processed in parallel to those procured for microarray studies, but retained and fixed in 1% glutaraldehyde. These cells were then concentrated and the pellet used to generate a cell block, which was processed, sectioned and then stained for microscopic examination. From this same colectomy specimen, a small strip of colonic mucosa was also procured, fixed, processed and stained for comparative purposes to document cellular heterogeneity (figure 6).


Normal colon epithelium: a dataset for the analysis of gene expression and alternative splicing events in colon disease.

Mojica W, Hawthorn L - BMC Genomics (2010)

Histology of colonic mucosa. (1a) Section of mucosa was stripped off the colectomy specimen and processed by formalin fixation and paraffin embedding. The mucosa is composed of the lamina propria and epithelium. As is evident, it is not a homogeneous collection of cells, but rather a composite of the cells that are present in the lamina propria (chronic inflammatory cells, smooth muscle cells and vessels) and epithelium (hematoxylin and eosin 10×). (1b) Histologic evidence of enrichment for colonic epithelial cells from the procurement approach described in the text (hematoxylin and eosin 20×).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2823691&req=5

Figure 6: Histology of colonic mucosa. (1a) Section of mucosa was stripped off the colectomy specimen and processed by formalin fixation and paraffin embedding. The mucosa is composed of the lamina propria and epithelium. As is evident, it is not a homogeneous collection of cells, but rather a composite of the cells that are present in the lamina propria (chronic inflammatory cells, smooth muscle cells and vessels) and epithelium (hematoxylin and eosin 10×). (1b) Histologic evidence of enrichment for colonic epithelial cells from the procurement approach described in the text (hematoxylin and eosin 20×).
Mentions: To control for variables associated with specimen integrity and sample processing, the time intervals between receipt of the sample in the operating suite and extirpation of each specimen was recorded. Additionally, the time intervals for delivery to the laboratory, placement of exfoliated cells in PBS with DTT, and the endpoint wherein enriched cells were stabilized by snap freezing were also documented and presented in table 3. A portion of the exfoliated cells from one case were processed in parallel to those procured for microarray studies, but retained and fixed in 1% glutaraldehyde. These cells were then concentrated and the pellet used to generate a cell block, which was processed, sectioned and then stained for microscopic examination. From this same colectomy specimen, a small strip of colonic mucosa was also procured, fixed, processed and stained for comparative purposes to document cellular heterogeneity (figure 6).

Bottom Line: One of the main objectives of this study was to generate a reference gene expression data set for normal colonic epithelium which can be used in comparisons with diseased tissues, as well as to provide a dataset that could be used as a baseline for studies in alternative splicing.For demonstration purposes, we have compared the data derived from these cells to a publically available set of tumor and matched normal colon data.Our analysis of splice variants illustrate that this is a very labor intensive procedure, requiring vigilant examination of the data.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular Oncology Program, Medical College of Georgia Cancer Center, Augusta, GA, USA.

ABSTRACT

Background: Studies using microarray analysis of colorectal cancer have been generally beleaguered by the lack of a normal cell population of the same lineage as the tumor cell. One of the main objectives of this study was to generate a reference gene expression data set for normal colonic epithelium which can be used in comparisons with diseased tissues, as well as to provide a dataset that could be used as a baseline for studies in alternative splicing.

Results: We present a dependable expression reference data set for non-neoplastic colonic epithelial cells. An enriched population of fresh colon epithelial cells were obtained from non-neoplastic, colectomy specimens and analyzed using Affymetrix GeneChip EXON 1.0 ST arrays. For demonstration purposes, we have compared the data derived from these cells to a publically available set of tumor and matched normal colon data. This analysis allowed an assessment of global gene expression alterations and demonstrated that adjacent normal tissues, with a high degree of cellular heterogeneity, are not always representative of normal cells for comparison to tumors which arise from the colon epithelium. We also examined alternative splicing events in tumors compared to normal colon epithelial cells.

Conclusions: The findings from this study represent the first comprehensive expression profile for non-neoplastic colonic epithelial cells reported. Our analysis of splice variants illustrate that this is a very labor intensive procedure, requiring vigilant examination of the data. It is projected that the contribution of this set of data derived from pure colonic epithelial cells will enhance studies in colon-related disease and offer a vital baseline for studies aimed at elucidating the mechanisms of alternative splicing.

Show MeSH
Related in: MedlinePlus