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Expression profiling of ductal carcinoma in situ by laser capture microdissection and high-density oligonucleotide arrays.

ABSTRACT: Gene expression profiling through the use of nucleic acid arrays is a powerful method for the molecular classification of human neoplasms. Laser capture microdissection is an equally useful technique to selectively isolate defined cell populations from heterogeneous histological tissue sections. In this report, we demonstrate how a modest use of laser capture microdissection is sufficient to isolate nanogram quantities of high-quality RNA. Together with the use of several internal standards and microcapillary electrophoresis of input RNA, two rounds of linear molecular amplification have been used to generate sufficient quantities of labeled target for hybridization to high-density oligonucleotide expression arrays. Results demonstrate that the technique is reproducible, generates only modest biasing of the original transcript population, and is comparable to the sensitivity achieved with standard methodology. Using this approach, we have compared the expression profiles of nonmalignant human breast epithelium and adjacent ductal carcinoma in situ lesions from breast cancer patients. Several genes, previously implicated in human breast cancer progression, demonstrate differential expression among the microdissected cell populations.

SUBMITTER: Luzzi V

PROVIDER: S-EPMC1891975 | biostudies-literature | 2001 Jun

REPOSITORIES: biostudies-literature

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Expression profiling of ductal carcinoma in situ by laser capture microdissection and high-density oligonucleotide arrays.

Luzzi V V Holtschlag V V Watson M A MA

The American journal of pathology 20010601 6

Gene expression profiling through the use of nucleic acid arrays is a powerful method for the molecular classification of human neoplasms. Laser capture microdissection is an equally useful technique to selectively isolate defined cell populations from heterogeneous histological tissue sections. In this report, we demonstrate how a modest use of laser capture microdissection is sufficient to isolate nanogram quantities of high-quality RNA. Together with the use of several internal standards and ...[more]

PMID: 11395378

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Project description:The etiology of Type 1 Diabetes (T1D) remains elusive. Enzymatically isolated and cultured (EIC) islets cannot fully reflect the natural protein composition and disease process of in vivo islets, because of the stress from isolation procedures. In order to study islet protein composition in conditions close to the natural environment, we performed proteomic analysis of EIC islets, and laser capture microdissected (LCM) human islets and acinar tissue from fresh-frozen pancreas sections of three cadaveric donors. 1104 and 706 proteins were identified from 6 islets equivalents (IEQ) of LCM islets and acinar tissue, respectively. The proteomic profiles of LCM islets were reproducible within and among cadaveric donors. The endocrine hormones were only detected in LCM islets, whereas catalytic enzymes were significantly enriched in acinar tissue. Furthermore, high overlap (984 proteins) and similar function distribution were found between LCM and EIC islets proteomes, except that EIC islets had more acinar contaminants and stress-related signal transducer activity proteins. The comparison among LCM islets, LCM acinar tissue and EIC islets proteomes indicates that LCM combined with proteomic methods enables accurate and unbiased profiling of islet proteome from frozen pancreata. This paves the way for proteomic studies on human islets during the progression of T1D.The etiological agent triggering autoimmunity against beta cells in Type 1 diabetes (T1D) remains obscure. The in vitro models available (enzymatically isolated and cultured islets, EIC islets) do not accurately reflect what happens in vivo due to lack of the natural environment where islets exist and the preparation-induced changes in cell physiology. The importance of this study is that we investigated the feasibility of laser capture microdissection (LCM) for the isolation of intact islets from frozen cadaveric pancreatic tissue sections. We compared the protein profile of LCM islets (9 replicates from 3 cadaveric donors) with that of both LCM acinar tissues (6 replicates from the same 3 cadaveric donor as LCM islets) and EIC islets (at least 4 replicates for each sample with the same islets equivalents) by using proteomics techniques with advanced instrumentation, nanoLC-Q Exactive HF Orbitrap mass spectrometry (nano LC-MS/MS). The results demonstrate that the LCM method is reliable in isolating islets with an intact environment. LCM-based islet proteomics is a feasible approach to obtain good proteome coverage for assessing the pathology of T1D using cadaveric pancreatic samples, even from very small sample amounts. Future applications of this LCM-based proteomic method may help us understand the pathogenesis of T1D and identify potential biomarkers for T1D diagnosis at an early stage.

Dataset Information

Expression profiling of ductal carcinoma in situ by laser capture microdissection and high-density oligonucleotide arrays.

Publications

Expression profiling of ductal carcinoma in situ by laser capture microdissection and high-density oligonucleotide arrays.

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