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PubMed2003

High quality RNA retrieved from samples obtained by using LMPC (laser microdissection and pressure catapulting) technology.

Isolation of intact RNA in high quality is the first and often the most critical step in performing many fundamental molecular biology experiments, and is essential for many techniques used in gene expression analysis. As many factors influence nucleic acid preservation, RNA isolation should include some important steps before and after the actual RNA extraction. We tested the influence of fixation and staining protocols regarding RNA integrity and concentration. A factor that is often underestimated is the absolute necessity for homogenous starting materials. Application of the LMPC technology allows for a rapidand highly precise procurement of purified cell populations suitable for a variety of downstream analyses.

Animals↗
PubMed1969

Proteolytic microdissection of smooth-surfaced vesicles of liver microsomes.

Digestion of rabbit liver microsomal smooth vesicles with Bacillus subtilis protease released proteins and peptide fragments from the vesicles, without solubilizing phospholipids and cholesterol. The proteolysis was, however, limited when about 30% of the protein had been solubilized. The same limitation was observed when the vesicles were treated with trypsin, chymotrypsin, or their combinations with the bacterial protease. The limited proteolysis was accompanied by selective solubilization of cytochrome b(5) and microsomal NADPH-specific flavoprotein, leaving the CO-binding hemoprotein and some other enzymes still attached to the vesicular membranes. Sucrose density gradient centrifugation of protease-treated vesicles indicated that all the vesicles had been attacked by the protease to similar extents. The behavior of intact and digested vesicles in dextran density gradient centrifugation suggested that the vesicles, even after proteolytic digestion, existed in the form of closed sacs which were impermeable to macromolecules such as dextran and proteases. It was concluded that only the outside surface of the vesicles is susceptible to the proteolytic action and that cytochrome b(5) and the NADPH-specific flavoprotein are located in the susceptible area.

Animals↗
PubMed2001

p53 missense mutations in microdissected high-grade ductal carcinoma in situ of the breast.

BACKGROUND: To understand the role of sporadic mutations in the tumor suppressor gene p53 (also known as TP53) in the pathogenesis of breast cancer, it is important to identify at which histologic stage such mutations first occur. We previously showed that a p53 mutation present in invasive breast cancer was found in all surrounding areas of ductal carcinoma in situ (DCIS) but not in areas of hyperplasia or normal breast epithelium. In the present investigation, we studied patients with DCIS, but without invasive breast cancer, to determine the spectrum of DCIS types that can harbor a p53 mutation. METHODS: Formalin-fixed, paraffin-embedded tissues from 94 patients with DCIS were evaluated histologically for the predominant cellular architectural pattern, degree of necrosis, and nuclear grade. Each specimen was also assigned an overall histologic grade (with the use of the Van Nuys Prognostic Index pathologic classification). Tissue specimens were stained immunohistochemically with an anti-p53 antibody. Positively stained tissue areas were analyzed for the presence of p53 mutations by single-strand conformation polymorphism and direct sequencing. All statistical tests were two-sided. RESULTS: DCIS from 10 of 94 patients were found to contain p53 missense mutations. All 10 were of a solid or a comedo histologic pattern and contained cells of nuclear grade 2 or 3 (i.e., more abnormal nuclei). The frequency of p53 missense mutations was statistically significantly different among the three overall histologic grade categories (zero [0%] of 49 with low-grade DCIS, one [4.35%] of 23 with intermediate-grade DCIS, and nine [40.9%] of 22 with high-grade DCIS; df = 2 and P<.0001). CONCLUSION: The DCIS types in patients in this series are representative of clinically detected DCIS. Our finding that p53 mutations can occur before the development of invasive breast cancer, particularly in DCIS of high histologic grade, has potentially important implications for prevention and treatment.

Breast Neoplasms↗
PubMed1989

PCR amplification of DNA microdissected from a single polytene chromosome band: a comparison with conventional microcloning.

A novel alternative to microcloning for the production of region specific chromosomal DNA is described. In this method, 'microamplification', single bands are dissected from polytene chromosomes and digested with Sau3A. Oligonucleotide adaptors are ligated to these fragments to provide convenient priming sites for polymerase chain reaction amplification. In this way, as much as 1 microgram of DNA can be amplified from a single band. Probes made from PCR amplified DNA from two such dissections have been used to probe cloned DNA form a 100 kb chromosome walk. Whereas conventional microcloning has generated cloned EcoRI fragments corresponding to 3-4 kb of the walk, the PCR probes cover greater than 90% of this chromosomal region. Thus microamplification is significantly more effective than microcloning in providing probes for establishing chromosomal walks.

Animals↗
PubMed2002

Sequential development of Hodgkin's disease and CD30+ diffuse large B-cell lymphoma in a patient with MALT-type lymphoma: evidence of different clonal origin of single microdissected Reed-Sternberg cells.

We observed in the same patient the development of a tonsil mucosa-associated lymphoid tissue-type lymphoma in 1994, a mediastinal Hodgkin's disease in 1998, and a colonic CD30+ anaplastic diffuse large B-cell lymphoma in 2000. A same-sized FR3-JH fragment was demonstrated by polymerase chain reaction, both at the level of total DNA and of single micromanipulated cells, showing monocytoid, Reed-Sternberg, or anaplastic morphology. Moreover, an identical IgH nucleotide sequence was detected in mucosa-associated lymphoid tissue-type lymphoma and colonic CD30+ anaplastic diffuse large B-cell lymphoma, whereas mediastinal Hodgkin's disease IgH rearrangement involved different VH and JH genes. CD30+ Reed-Sternberg and diffuse large B-cell lymphoma cells contained Epstein-Barr virus EBER sequences that were not observed at the level of mucosa-associated lymphoid tissue-type lymphoma. Therefore, Epstein-Barr virus infection may have played a role in diffuse large B-cell lymphoma transformation of mucosa-associated lymphoid tissue-type lymphoma and in the lymphomagenesis of Hodgkin's disease. In addition to their different clonal origin, Reed-Sternberg cells of Hodgkin's disease expressed a CD15+, CD20+ (rare cells), CD30+, Oct-2-, EBNA2-, LMP1+ phenotype, whereas anaplastic and Reed-Sternberg-like cells of diffuse large B-cell lymphoma were CD15-, CD20+, CD30+, Oct-2+, EBNA2+, and LMP1+. Interestingly, we also detected scattered CD30+ Epstein-Barr virus- large cells with prominent nucleoli in the initial tonsil mucosa-associated lymphoid tissue-type lymphoma, suggesting that these cells could be prone to Epstein-Barr virus infection and/or large cell transformation.

DNA, Neoplasm↗