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At least 613 records · Page 34Linked to original sources
PubMed2001

Molecular genetic evidence for different clonal origin of components of human renal angiomyolipomas.

Renal angiomyolipoma is a benign neoplasm composed of variable proportions of blood vessels, smooth muscle, and adipose tissue. Smooth muscle, adipose tissue, blood vessels, and adjacent normal kidney tissue were separately microdissected from sections prepared from formalin-fixed, paraffin-processed tissues from angiomyolipomas from 18 women. X chromosome inactivation analysis using the methylation pattern at exon 1 of the human androgen receptor gene on chromosome Xq11-12 was used to study the clonal origin of each component. Nonrandom inactivation of X chromosomes was found in six of the 15 informative tumors. The smooth muscle and adipose tissue showed differing patterns of nonrandom inactivation of X chromosomes in five angiomyolipomas and the same pattern of nonrandom inactivation of X chromosomes in one. Samples from the blood vessels showed random inactivation of X chromosomes in all informative cases. Our data showed that the adipose tissue and smooth muscle cells of renal angiomyolipoma are both monoclonal but may arise independently. The coexistence of tumor subclones with morphologic heterogeneity can lead to the formation of a clinically detectable tumor.

Adipose Tissue↗
PubMed2005

Clonality analysis for normal and cancerous colon tissues with human androgen receptor gene polymerase chain reaction.

We assessed the feasibility of clonality analysis with human androgen receptor gene polymerase chain reaction in terms of the sensitivity and specificity for normal and cancerous colonic tissues taken from fourteen informative cases selected from 22 women with colonic adenocarcinoma. Ten crypts microdissected from each 10-microm-thick cryostat sections and whole tissues were used as samples. DNA was extracted from the samples and amplified with and without prior enzyme digestion. These products were analyzed by capillary electrophoresis for clonality. Of the whole-tissue DNA, none of the normal tissues and seven (50.0%) of the cancerous tissues showed monoclonality. Of the microdissected samples, monoclonality was found in 88.4% (107/121) of normal crypts and 95.9% (117/122) of cancerous crypts. Samples composed of crypts with short and long alleles were found in eight of the 14 normal colonic mucosae, but in none of the cancerous tissues. We concluded that the sensitivity of this method is limited for both whole-tissue DNA and microdissected-tissue DNA, because monoclonality from small samples does not always indicate monoclonality of the entire lesion. The high specificity of this method, however, allows polyclonal results in whole tissues to be confirmed by additional analysis of microdissected tissues.

Adenocarcinoma↗
PubMed1993

Neuroendocrine neoplasms of the lung are not associated with point mutations at codon 12 of the Ki-ras gene.

The most prominent abnormality of ras proto-oncogenes in human lung tumours has involved point mutations at codon 12 of the Ki-ras gene. We have analysed 35 tumour samples of neuroendocrine lung neoplasms (ten carcinoid tumours, ten well-differentiated neuroendocrine carcinomas, and 15 intermediate/small cell neuroendocrine carcinomas) for a point mutation at this site. For this purpose, formalin-fixed and paraffin-embedded tissue sections were microdissected to remove non-tumours areas. DNA in the remaining tumour tissue was amplified in vitro by the polymerase chain reaction (PCR) and double-stranded PCR products were subjected to sequence analysis. Neither point mutations at codon 12 nor additional structural alterations at codons 1-32 were detected in Ki-ras gene. Our results suggest that point mutations at codon 12 of the Ki-ras gene do not seem to be involved in the pathogenesis of pulmonary neuroendocrine neoplasms.

Base Sequence↗
PubMed2001

Laser-assisted microdissection of membrane-mounted paraffin sections for polymerase chain reaction analysis: identification of cell populations using immunohistochemistry and in situ hybridization.

Laser microbeam microdissection (LMM) is an increasingly important method for obtaining pure cell samples for genetic and proteomic analysis. Immunohistochemistry (IHC) and in situ hybridization (ISH) are useful techniques for targeting specific cell populations for microdissection but are difficult to apply with the tissue support membranes often used during LMM. Using detection of cytokeratins and Epstein-Barr virus gene products in head and neck carcinoma as a model, we describe optimized protocols for membrane and section preparation and for low temperature antigen retrieval that allow IHC and ISH to be used reliably on membrane mounted paraffin tissue sections. Visualization of cellular targets was markedly improved by staining and this could be further improved using a variety of optical media before microdissection. Tissue fragments thus stained were suitable for subsequent polymerase chain reaction analysis of extracted DNA using standard techniques. These IHC and ISH procedures are generally applicable and will be useful for detecting a wide range of antigens and nucleic acids in paraffin sections in conjunction with LMM.

Head and Neck Neoplasms↗
PubMed1982

Juxtaglomerular angiotensin II formation.

Previous studies have indicated the existence of an unknown AII-forming enzyme (AFE) in the glomerular region. This investigation was undertaken to characterize this enzyme further and to elucidate its possible physiologic role. AFE activity was found in cortical tissue and in microdissected superficial and juxtamedullary glomeruli but not in renal medulla. It could be blocked by the converting enzyme (CE) inhibitors SQ 20881 (133.5 micrograms/ml; 70%), SQ 14225 (20 ng/ml; 80%), and DFP (20 micrograms/ml; 80%). In contrast to AI CE, AFE was neither blocked by EDTA nor stimulated by sodium chloride. In a further study, AFE activity and AII immunoreactivity were measured simultaneously in different renal structures and in plasma under control conditions and following water deprivation. Highest AII immunoreactivity was demonstrated in microdissected glomeruli (about 10 ng/g; N = 11) and lower amounts in cortical tissue (2.83 +/- 0.72 ng/g; N = 12), avascular cortical tissue (1.80 +/- 1.08 ng/g; N = 3), outer medulla (1.52 +/- 0.33 ng/g; N = 12), inner medulla (0.72 +/- 0.17 ng/g; N = 2), and in plasma (0.212 +/- 0.066 ng/ml; N = 12). Water deprivation had no measurable effect on AFE activity; but AII immunoreactivity increased significantly in all samples. This indicates that, in spite of the extremely high juxtaglomerular renin activity, AFE cannot be regarded as the limiting enzyme for local AII formation under the conditions investigated.

Angiotensin II↗
PubMed2006

Methylation of p16 and Ras association domain family protein 1a during colorectal malignant transformation.

Accurate assessment of gene methylation in formalin-fixed, paraffin-embedded archived tissue (FF-PEAT) by microdissection remains challenging because the tissue volume is small and DNA is damaged. In addition, methods for methylation assessment, such as methylation-specific PCR (MSP), require sodium bisulfite modification (SBM) on purified DNA, which causes major loss of DNA. On-slide SBM, in which DNA is modified in situ before isolation of tumor cells, eliminates DNA purification steps and allows histology-oriented assessment of gene methylation. This study describes a protocol and use of on-slide SBM using 20 FF-PEAT of colorectal cancers with intratumoral adenoma components to detect accumulation of gene methylation during colorectal malignant transformation. Deparaffinized tissue sections were incubated in sodium bisulfite solution for 8 hours at 60 degrees C, stained with hematoxylin, and then microdissected. Proteinase K lysate was directly used as a template in subsequent PCR. Using on-slide SBM, 282-bp-long bisulfite direct sequencing was possible. Yield of modified DNA was 2.6-fold greater than standard SBM on average. The mean conversion rate was 97%, and false-positive or false-negative results were not observed in subsequent MSP. Intratumoral heterogeneity by accumulation of p16 and Ras association domain family protein 1a methylation during malignant transformation were shown by MSP comparing cancer with adenoma parts within a single section. On-slide SBM is applicable in most methylation studies using FF-PEAT. It allows detailed, intratumoral analysis of methylation heterogeneity within solid tumors. On-slide SBM will significantly improve our approach and understanding of epigenetic events in minimal disease and the carcinogenic process.

Alu Elements↗
PubMed2004

Laser-assisted microdissection of the kidney: fundamentals and applications.

Laser-assisted microdissection (LAM) permits the procurement of relatively pure cell populations from histological sections. When applied to the kidney, LAM combined with molecular biological techniques has expanded our understanding of renal biology and pathology. Both frozen and fixed renal tissues can be microdissected. However, sample type and tissue processing can influence the quality of molecular data generated. Data analysis may also be complicated by relative variations in gene expression levels. Importantly, preliminary studies have shown that molecular data obtained following LAM on the kidney can offer new diagnostic and prognostic information. Thus, LAM and molecular markers may eventually become incorporated into the routine kidney biopsy examination.

Animals↗
PubMed2001

High rates of loss of heterozygosity on chromosome 19p13 in human breast cancer.

We have recently discovered that the nuclear matrix protein SAFB is an oestrogen receptor corepressor. Since it has become clear that many steroid receptor cofactors play important roles in breast tumorigenesis, we investigated whether SAFB could also be involved in breast cancer. To address this question, the gene locus was examined for structural alterations in breast cancer tissue. Laser capture microdissection was used for isolating DNA from paired primary breast tumour and normal tissue specimens, and the loss of heterozygosity (LOH) at chromosome 19p13.2-3 was determined by use of microsatellite markers. LOH was detected at the marker D19S216, which colocalizes with the SAFB locus, in specimens from 29 (78.4%) of 37 informative patients. The peak LOH rate occurred at D19S216 near the SAFB locus, with LOH frequencies ranging from 21.6% to 47.2% at other markers. The finding of a very high LOH rate at the marker D19S216 strongly indicates the presence of a breast tumour-suppressor gene locus. While preliminary findings of mutations in SAFB suggest that this indeed may be a promising candidate, other potential candidate genes are located at this locus.

Antigens, Nuclear↗
PubMed2004

Novel proteomic approaches for tissue analysis.

Proteomics, the global study of protein expression and characteristics, has recently emerged as a key component in the field of molecular analysis. The dynamic nature of proteins, from ion channels to chaperones, presents a challenge, yet the understanding of these molecules provides a rich source of information. When applying proteomic analysis directly to human tissue samples, additional difficulties arise. The following article presents an overview of the current proteomic tools used in the analysis of tissues, beginning with conventional methods such as western blot analysis and 2D polyacrylamide gel electrophoresis. The most current high-throughput techniques being used today are also reviewed. These include protein arrays, reverse-phase protein lysate arrays, matrix-assisted laser desorption/ionization, surface-enhanced laser desorption/ionization and layered expression scanning. In addition, bioinformatics as well as issues regarding tissue preservation and microdissection to obtain pure cell populations are included. Finally, future directions of the tissue proteomics field are discussed.

Animals↗
PubMed2003

[Gene expression analysis in liver tissue at a single cell level by nested polymerase chain reaction and laser microdissection].

OBJECTIVE: To investigate the measurements of gene expressing at a single hepatocyte level. METHODS: Individual hepatocyte was isolated from cryostat tissue section using laser microdissection technique. To detect the mRNA expressed by single hepatocyte, RNA was extracted, reversely transcribed to cDNA and amplified by nested polymerase chain reaction (PCR). RESULTS: Single cell was microdissected from cryostat tissue using an ultraviolet laser micromanipulator. The RNA could be extracted from the isolated cell(s), and the RT-PCR production could be observed after electrophoresis, whose quantitation was compatible with the number of cells. CONCLUSION: Combining laser microdissection and nested RT-PCR can monitor gene expression at a single cell level in vivo.

Dissection↗
PubMed2002

Microdissection techniques for cancer analysis.

One difficulty in studying molecular changes of tumours has been the inability to isolate DNA and RNA from a homogeneous cell population. The combination of several new technologies should help overcome these hurdles. Microdissection is a technique for rapid and easy procurement of a pure cellular subpopulation away from its complex tissue milieu. Laser-assisted microdissection has recently been identified as a quick, simple and effective method by which microdissection of complex tissue specimens can be routinely performed for molecular analysis. With the advent of laser microdissection, cDNA libraries can be developed from pure cells obtained directly from stained neoplastic tissue, and microarrays of thousands of genes can now be used to examine gene expression in microdissected tumour tissue samples. This review will concentrate on the application of different microdissection techniques in the area of cancer research.

Cell Separation↗
PubMed2002

Osteopontin as a potential diagnostic biomarker for ovarian cancer.

CONTEXT: Development of new biomarkers for ovarian cancer is needed for early detection and disease monitoring. Analyses involving complementary DNA (cDNA) microarray data can be used to identify up-regulated genes in cancer cells, whose products may then be further validated as potential biomarkers. OBJECTIVE: To describe validation studies of an up-regulated gene known as osteopontin, previously identified using a cDNA microarray system. DESIGN, SETTING, AND PARTICIPANTS: Experimental and cross-sectional studies were conducted involving ovarian cancer and healthy human ovarian surface epithelial cell lines and cultures, archival paraffin-embedded ovarian tissue collected between June 1992 and June 2001, and fresh tissue and preoperative plasma from 144 patients evaluated for a pelvic mass between June 1992 and June 2001 in gynecologic oncology services at 2 US academic institutions. Plasma samples from 107 women selected from an epidemiologic study of ovarian cancer initiated between May 1992 and March 1997 were used as healthy controls. MAIN OUTCOME MEASURES: Relative messenger RNA expression in cancer cells and fresh ovarian tissue, measured by real-time polymerase chain reaction as 2(-DeltaDeltaCT)(a quantitative value representing the amount of osteopontin expression); osteopontin production, localized and scored in ovarian healthy and tumor tissue with immunohistochemical studies; and amount of osteopontin in patient vs control plasma, measured using an enzyme-linked immunoassay. RESULTS: The geometric mean for 2(-DeltaDeltaCT)for osteopontin expression in 5 healthy ovarian epithelial cell cultures was 4.1 compared with 270.4 in 14 ovarian cancer cell lines (P =.03). The geometric mean 2(-DeltaDeltaCT)for osteopontin expression in tissue from 2 healthy ovarian epithelial samples was 9.0 compared with 164.0 in 27 microdissected ovarian tumor tissue samples (P =.06). Immunolocalization of osteopontin showed that tissue samples from 61 patients with invasive ovarian cancer and 29 patients with borderline ovarian tumors expressed higher levels of osteopontin than tissue samples from 6 patients with benign tumors and samples of healthy ovarian epithelium from 3 patients (P =.03). Osteopontin levels in plasma were significantly higher (P<.001) in 51 patients with epithelial ovarian cancer (486.5 ng/mL) compared with those of 107 healthy controls (147.1 ng/mL), 46 patients with benign ovarian disease (254.4 ng/mL), and 47 patients with other gynecologic cancers (260.9 ng/mL). CONCLUSIONS: Our findings provide evidence for an association between levels of a biomarker, osteopontin, and ovarian cancer and suggest that future research assessing its clinical usefulness would be worthwhile.

Biomarkers, Tumor↗
PubMed1994

Multiple PCR analyses on trace amounts of DNA extracted from fresh and paraffin wax embedded tissues after random hexamer primer PCR amplification.

AIM: To establish a simple and reliable polymerase chain reaction (PCR) methodology for random amplification of whole genomic DNA from limited histopathological samples. METHODS: Trace amounts of genomic DNA extracted from fresh tissue and individual lymphoid follicles microdissected from archival paraffin wax tissue sections were amplified using a two-phase PCR protocol with random hexamers as primers (RP-PCR). The randomly amplified DNA samples were used as templates for specific PCR amplifications. To check the fidelity of the RP-PCR, products of the specific PCR amplifications were further analysed by single stranded conformation polymorphism (SSCP) or sequencing. RESULTS: Using a minute fraction of RP-PCR template pool, multiple PCR analyses, including those for beta globin gene, p53 gene (exon 5-6, exon 7, exon 8-9 and exon 7-9), and rearranged immunoglobulin heavy chain gene fragments (VH framework 3 to JH and VH framework 2 to JH) were successfully performed. No artefactual mutations were identified in the products of these specific PCR reactions by SSCP or sequencing when compared with the products from the original DNA. CONCLUSION: This method is simple and reliable, and permits multiple genetic analyses when only a limited amount of tissue is available.

Base Sequence↗
PubMed2000

Quantitation of DNA extracted after micropreparation of cells from frozen and formalin-fixed tissue sections.

Quantitation of DNA from microdissected fresh-frozen or paraffin-embedded tissue sections would be not only a valuable tool for ensuring optimum reaction conditions for many types of qualitative polymerase chain reaction (PCR) analyses, but also a prerequisite for any kind of subsequently performed genetic analyses aimed at the absolute quantitation of target sequences. The present study describes the quantitation of DNA after microdissection and extraction of cells with the PicoGreen fluorescence method. The limits of detection and of quantitative determination, respectively, have been determined by measuring dilutional series of three different DNA extractions, using either a medium-scale preparation from a solid tissue specimen or a known number of leukocytes or microdissected cells from frozen tumor sections. As corresponding limits of detection, 26, 24, and about 40 diploid genomes, and as limits of quantitative determination, 80, 73, and about 120 diploid genomes were obtained. Furthermore, it was shown that formalin fixation as well as hematoxylin staining of frozen sections with Delafield's and Mayer's alum or Weigert's iron hematoxylin before microdissection significantly diminishes the amount of extractable DNA and may lead to less reliable results, even of qualitative PCR analysis. In conclusion, the PicoGreen method allows precise quantitation of DNA corresponding to a minimum of about 120 diploid cells. It provides the basis for reliable qualitative analyses as well as the precondition for further quantitative genetic measurements from microdissected frozen or formalin-fixed and paraffin-embedded tissue sections.

DNA, Neoplasm↗
PubMed2000

IF-LCM: laser capture microdissection of immunofluorescently defined cells for mRNA analysis rapid communication.

BACKGROUND: The next phase of the molecular revolution will bring functional genomics down to the level of individual cells in a tissue. Laser capture microdissection (LCM) coupled with reverse transcription-polymerase chain reaction (RT-PCR) can measure gene expression in normal, cancerous, injured, or fibrotic tissue. Nevertheless, targeting of specific cells may be difficult using routine morphologic stains. Immunohistochemistry can identify cells with specific antigens; however, exposure to aqueous solutions destroys 99% of the mRNA. Consequently, there is an overwhelming need to identify specific tissue cells for LCM without mRNA loss. We report on a rapid immunofluorescent LCM (IF-LCM) procedure that allows targeted analysis of gene expression. METHODS: A LCM microscope was outfitted for epifluorescence and light level video microscopy. Heat filters were added to shield the image intensifier from the laser. Frozen sections were fluorescently labeled by a rapid one minute incubation with anti-Tamm-Horsfall antibody and an ALEXA-linked secondary antibody. Fluorescently labeled thick ascending limb (TAL) cells were detected by low light level video microscopy, captured by LCM, and mRNA was analyzed by RT-PCR for basic amino acid transporter, Tamm-Horsfall protein, and aquaporin-2. RESULTS: The immunofluorescently identified TAL could be cleanly microdissected without contamination from surrounding tubules. The recovery of RNA following rapid immunofluorescence staining was similar to that obtained following hematoxylin and eosin staining, as assessed by RT-PCR for malate dehydrogenase. CONCLUSIONS: We conclude that the new apparatus and method for the immunofluorescent labeling of tissue cells targeted for LCM can isolate pure populations of targeted cells from a sea of surrounding cells with highly acceptable preservation of mRNA. Since the TAL is minimally injured following ischemia, identification of the different responses between TAL and surrounding tissue in damaged kidneys may provide new therapeutic targets or agents for the treatment of acute renal failure.

Acridine Orange↗
PubMed2001

Quantitative amplification of genomic DNA from histological tissue sections after staining with nuclear dyes and laser capture microdissection.

Laser capture microdissection (LCM) allows the selective sampling of tissue from histological sections. A prerequisite for this technique is the availability of histological dyes that do not interfere with downstream analysis of the sampled genetic material. We have examined the effect of four histological nuclear dyes (methyl green, hematoxylin, toluidine blue O, azure B) on TaqMan polymerase chain reaction amplification of beta-actin genomic DNA prepared from fixed and frozen tissue. Tissue sampled from the histological sections by manual dissection was compared with tissue sampled by LCM. As previously reported, when manually dissected tissue sections were analyzed, polymerase chain reaction amplification of DNA after hematoxylin staining was inferior to that after staining with the other dyes. In contrast, when tissue sampled by LCM was examined, DNA recovery after hematoxylin staining was equivalent to the recovery after methyl green staining. We conclude that DNA recovery from LCM-sampled tissue is independent of the histological stain chosen to highlight nuclear detail.

Cell Nucleus↗