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Results for “Microdissection”

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At least 73 records · Page 4Linked to original sources

PubMed2004

The value of polymerase chain reaction detection of Mycobacterium tuberculosis in granulomas isolated by laser capture microdissection.

AIMS: The aim of this study was to investigate the usefulness of polymerase chain reaction (PCR) detection of Mycobacterium tuberculosis in granulomas isolated by laser capture microdissection (LCM). METHODS: The PCR DNA amplification method was used to detect M. tuberculosis in granulomas microdissected from one section stained by haematoxylin and eosin (H&E) from a formalin-fixed paraffin-embedded specimen. The results were compared to those obtained from PCR performed from 10 whole paraffin sections of 5 micro m each, and with the histology, culture and the patient's clinical findings. RESULTS: Forty-nine formalin-fixed and paraffin-embedded samples from 49 patients with a histological suspicion of a mycobacterial infection were investigated. Using culture as the reference method, the sensitivity for the detection of M. tuberculosis was 92% and the specificity was 100% using PCR from microdissected granulomas and were similar to those obtained by using PCR from 10 whole sections. CONCLUSIONS: The PCR method of examination of microdissected granulomas from deparaffinised sections is a sensitive, specific and rapid method for the detection of M. tuberculosis in formalin-fixed and paraffin-embedded samples. The method is as sensitive as that using PCR on 10 whole tissue sections, thus making it suitable for small biopsies. However, although these methods reduce the delay in diagnosis, culture remains the gold standard for identification of mycobacteria in tissue. Culture also allows for the testing of antibiotic sensitivity of any isolated species, in this way determining appropriate treatment.

Animals↗

PubMed2004

Assessment of gene expression in head and neck carcinoma using laser capture microdissection and real-time reverse transcription polymerase chain reaction.

OBJECTIVES: To quantify gene expression in tumor cells from human head and neck squamous cell carcinomas (HNSCC) using laser capture microdissection (LCM). STUDY DESIGN: Histopathologically identified HNSCC cells were microdissected from frozen sections, RNA was isolated, and vascular endothelial growth factor (VEGF) gene expression was measured by real-time reverse transcriptase polymerase chain reaction (RT-PCR). MATERIALS AND METHODS: Two human HNSCC tumor samples and matched normal mucosal biopsies and five human xenograft tumor specimens were harvested, embedded, and frozen in OCT. The frozen tumors were sectioned to 8 to 10 mum in thickness, and hematoxylin-eosin (H&E) staining was performed before LCM. An estimated 2,000 to 3,000 tumor cells were microdissected from frozen sections and processed for RNA isolation. mRNA for VEGF was analyzed by real time RT-PCR (TaqMan) with commercially available primers and probes. RESULTS: Two thousand to 3000 cells were necessary to obtain a suitable quantity of RNA for subsequent gene expression study by real-time RT-PCR. The gene expression of VEGF, a major tumor angiogenic factor, was tested in microdissected HNSCC and compared with uninvolved normal mucosal controls. A greater than seven-fold increase of VEGF expression in tumor specimens versus mucosal controls was observed. CONCLUSIONS: LCM is a novel sample conserving technique that allows the precise selection of tumor cells from a heterogeneous architecture. The combination of LCM and real-time RT-PCR appears particularly efficacious for studying HNSCC molecular pathogenesis and identifying tissue-specific biomarkers.

Biopsy, Needle↗

PubMed2005

Methodology for preservation of high molecular-weight RNA in paraffin-embedded tissue: application for laser-capture microdissection.

Laser-capture microdissection techniques have enhanced the ability to perform molecular studies of pure-cell populations. Although many technical factors affect the outcome of the procedure, none is more critical than the appropriate handling of the tissue. Because extraction of intact RNA from paraffin-embedded tissue is a difficult and inconsistent process, frozen sections with their attendant problems are used for this purpose. The major limitation of frozen section is its inferior morphologic quality compared with paraffin-embedded sections that may complicate accurate identification of cells during microdissection. We have developed a procedure that provides both high-quality histomorphology and RNA preservation in paraffin-embedded tissue. It is based on the use of a methanol-based fixative coupled with microwave-assisted rapid tissue processing. This technology in conjunction with a modified hematoxylin-eosin stain and a RNA extraction method allows isolation of high molecular-weight RNA from laser-capture microdissected, hematoxylin and eosin-stained paraffin sections. The high quality of the extracted RNA was confirmed by capillary electrophoresis and RT-PCR. The combination of a methanol-based fixative, rapid microwave tissue processing, and a modified hematoxylin and eosin stain produces paraffin sections that yield high molecular-weight RNA upon microdissection. This methodology opens the door for a wide range of gene expression analyses using paraffin-embedded tissue.

Electrophoresis, Capillary↗

PubMed2005

Real-time polymerase chain reaction and laser capture microdissection for the diagnosis of BK virus infection in renal allografts.

We used real-time polymerase chain reaction (PCR) technology to detect BK virus (BKV) in H and E-stained kidney biopsy sections, using laser capture microdissection. Renal allograft biopsy specimens from 4 patients with the histopathologic diagnosis of BKV-associated nephropathy (BKVAN; group 1) and 3 patients suspected to have BKVAN but without diagnostic histologic features (group 2) were retrieved. Diagnostic inclusion-bearing cells were microdissected by laser capture microscopy from group 1. Renal tubular epithelial cells were microdissected randomly in group 2. DNA was extracted and real-time amplification performed using primers targeting the large "T" and small "t" regions of the BKV and JC virus genomes. Tubular epithelial cells from a case without evidence of BKV infection were used as negative controls in a similar reaction. BKV presence was demonstrated only in epithelial cells containing typical viral inclusions. Group 2 and negative control samples were confirmed as negative for BKVAN. Real-time PCR technology can be used to detect BKV in H and E-stained, paraffin-embedded tissue sections. This technique detected BKV in tubular epithelial cells of renal allografts. To our knowledge, this is the first report of detecting BKV in laser capture microdissected renal biopsy specimens using real-time PCR.

BK Virus↗

PubMedDate not supplied

Gene expression profiling of microdissected pancreatic ductal carcinomas using high-density DNA microarrays.

Pancreatic ductal adenocarcinoma (PDAC) remains an important cause of malignancy-related death and is the eighth most common cancer with the lowest overall 5-year relative survival rate. To identify new molecular markers and candidates for new therapeutic regimens, we investigated the gene expression profile of microdissected cells from 11 normal pancreatic ducts, 14 samples of PDAC, and 4 well-characterized pancreatic cancer cell lines using the Affymetrix U133 GeneChip set. RNA was extracted from microdissected samples and cell lines, amplified, and labeled using a repetitive in vitro transcription protocol. Differentially expressed genes were identified using the significance analysis of microarrays program. We found 616 differentially expressed genes. Within these, 140 were also identified in PDAC by others, such as Galectin-1, Galectin-3, and MT-SP2. We validated the differential expression of several genes (e.g., CENPF, MCM2, MCM7, RAMP, IRAK1, and PTTG1) in PDAC by immunohistochemistry and reverse transcription polymerase chain reaction. We present a whole genome expression study of microdissected tissues from PDAC, from microdissected normal ductal pancreatic cells and pancreatic cancer cell lines using high-density microarrays. Within the panel of genes, we identified novel differentially expressed genes, which have not been associated with the pathogenesis of PDAC before.

Aged↗

PubMed2005

Compartment-specific quantitative gene expression analysis after laser microdissection from archival renal allograft biopsies.

BACKGROUND: Various immunological and non-immunological pathomechanisms are responsible for the cellular damage in renal allografts. Since the kidney is an anatomically complex organ with functional and morphological heterogeneous compartments (interstitium, tubuli, vessels, glomeruli), the local response to injury maybe variable, therefore, the identification of local pathomechanisms is important. AIM: To elucidate any discrepancies in quantitative mRNA expression profiles between a total specimen analysis and a cell-specific evaluation after laser microdissection. METHODS: Real-time RT-PCR was performed for complement component C3 and heme oxygenase-1 (HO-1) genes compared to the housekeeping gene beta-actin using whole section RNA extracted from formalin-fixed and paraffin-embedded archival material of 16 explanted, rejected renal allografts. Ten non-transplant nephrectomies served as controls. For five cases from each group, five different compartments of the organs (interstitium, proximal tubuli, distal tubuli, vessels, glomeruli) were microdissected and quantitative analysis for C3 and HO-1 was performed identically. RESULTS: Whole section mRNA expression analysis: the data showed a constant expression of the housekeeping gene beta-actin, a 7-fold increased expression of C3 and a 3-fold decreased expression of HO-1 in the allograft group as compared to the control group. mRNA expression results from microdissected compartments: in the control group, C3 and HO-1 expression could only be detected in the proximal tubuli of all cases whereas all five compartments analyzed from the rejecting kidneys showed expression of the two genes. In the allografts, expression levels of the investigated genes varied considerably not only among the different compartments but between individual cases as well. CONCLUSION: Laser microdissection combined with real-time RT-PCR is a feasible approach for retrospective quantitative gene expression analysis in formalin-fixed and paraffin-embedded renal allograft specimens. As shown for C3 and HO-1, cell-specific expression patterns ofpathogenetically relevant genes vary considerably between individual cases. A close correlation of morphology and cell-specific gene expression analysis will contribute to the elucidation of the complex pathogenesis of chronic renal allograft nephropathy.

Actins↗

PubMed2003

[Manual microdissection of defined cells and RNA extraction for gene expression analysis of esophageal carcinoma progress].

OBJECTIVES: To isolate cells of interest from heterogeneous tissue blocks to obtain accurate representations of molecular alterations acquired by neoplastic cells so as to meet the demands of further study on gene expression patterns of the esophageal carcinoma (EC) evolution. METHODS: Blocks of EC were stored at -70 degrees C as close as possible to the time of surgical resection. The tissue block was embedded in OCT and frozen sections of 35 microns in thickness were cut in a cryostat under strict RNAse-free conditions. Individual frozen sections were mounted on plain glass slides and 30-gauge needle attached to a 1 ml syringe was used to microdissect defined cells in the sections. The procured cells were used for total RNA extraction. RESULTS: An optimized protocol of manual microdissection was developed successfully whereby regions with an area as small as 1/25 mm2 could be accurately dissected. The RNA recovered from procured cells was of high quality suitable for subsequent applications of molecular analysis as assessed of 18S and 28S rRNAs by electrophoresis on agarose gel. CONCLUSIONS: It is believed that manual microdissection is capable to procure defined cell populations from complex primary tissues, thus allowing investigation of tissue-, cell-, and function-specific gene expression patterns. The technique is simple, easy to perform, versatile, and of particular usefulness when laser capture microdissection (LCM) is practically unavailable.

Cell Separation↗

PubMed1996

Diagnosis of four chromosome abnormalities of unknown origin by chromosome microdissection and subsequent reverse and forward painting.

A molecular cytogenetic method consisting of chromosome microdissection and subsequent reverse/forward chromosome painting is a powerful tool to identify chromosome abnormalities of unknown origin. We present 4 cases of chromosome structural abnormalities whose origins were ascertained by this method. In one MCA/MR patient with an add(5q)chromosome, fluorescence in situ hybridization (FISH), using probes generated from a microdissected additional segment of the add(5q) chromosome and then from a distal region of normal chromosome 5, confirmed that the patient had a tandem duplication for a 5q35-qter segment. Similarly, we ascertained that an additional segment of an add(3p) chromosome in another MCA/MR patient had been derived from a 7q32-qter segment. In a woman with a history of successive spontaneous abortions and with a minute marker chromosome, painting using microdissected probes from the whole marker chromosome revealed that it was i(15)(p10) or psu dic(15;15)(q11;q11). Likewise, a marker observed in a fetus was a ring chromosome derived from the paracentromeric region of chromosome 19. We emphasize the value of the microdissection-based chromosome painting method in the identification of unknown chromosomes, especially for marker chromosomes. The method may contribute to a collection of data among patients with similar or identical chromosome abnormalities, which may lead to a better clinical syndrome delineation.

Abnormalities, Multiple↗

PubMed1999

Laser-assisted microdissection: applications in molecular pathology.

Tissue microdissection is potentially one of the most useful techniques in molecular pathology. Laser-assisted microdissection has been developed to procure precisely the cells of interest in a tissue specimen, in a rapid and practical manner. Together with multiplex molecular approaches, it is now feasible to study genetic alterations and isolate genes and proteins in defined cell populations from complex normal and diseased tissues. The fundamental advantage of this technique is the possibility of capturing single cells from which high-quality DNA and mRNA can be isolated for analysis of sequence and quantitation of expression. Moreover, the integration of laser-assisted microdissection and proteomic analysis could identify novel protein markers for disease. The advent of laser-assisted microdissection is likely to have a profound impact on molecular pathology.

Cell Separation↗

PubMed1989

Telomeric sequences derived from laser-microdissected polytene chromosomes.

Telomeric fragments from salivary gland squashes of Drosophila melanogaster Oregon R. were produced by a new microdissection technique, UV laser microbeam dissection. Microdissection, an essential step in microcloning procedures, is usually performed using micromanipulators and microneedles. Recently it has been shown that microdissection can be improved to very high precision if a laser coupled into a microscope is used. A laser microbeam, generated by an excimer pumped dye laser, allows chromosomes to be cut into slices of less than 0.5 micron. Here it is shown, that single copy DNA probes prepared from Drosophila chromosomes by laser microdissection and microcloning relocalize to the chromosomal regions from which they are derived. The combination of laser technique and microcloning provides an advantageous approach for rapid genetic analysis with potential for the study of genetic diseases and genome mapping.

Animals↗

PubMed2001

Tissue microdissection techniques in quantitative genome and gene expression analyses.

Current advances in quantitative genome and gene expression analyses allow precise molecular genetic fingerprinting of tumor tissues. A crucial factor for the reliability of the data obtained with these refined techniques is the use of morphologically well-defined cell populations. Microdissection technology has been developed to procure pure cell populations from specific areas of tissue sections under microscopic control. This review covers techniques of tissue microdissection in the context of commonly used methods of quantitative genome and gene expression analysis. The first part of the review will summarize the technical aspects of various methods developed for tissue microdissection. In the latter part, current applications of quantitative genome and gene expression analysis techniques employed in microdissected tissue samples will be described.

Animals↗

PubMed2003

Liver gene expression profiles of rats treated with clofibric acid: comparison of whole liver and laser capture microdissected liver.

Clofibric acid (CLO) is a peroxisome proliferator (PP) that acts through the peroxisome proliferator activated receptor alpha, leading to hepatocarcinogenesis in rodents. CLO-induced hepatocarcinogenesis is a multi-step process, first transforming normal liver cells into foci. The combination of laser capture microdissection (LCM) and genomics has the potential to provide expression profiles from such small cell clusters, giving an opportunity to understand the process of cancer development in response to PPs. To our knowledge, this is the first evaluation of the impact of the successive steps of LCM procedure on gene expression profiling by comparing profiles from LCM samples to those obtained with non-microdissected liver samples collected after a 1 month CLO treatment in the rat. We showed that hematoxylin and eosin (H&E) staining and laser microdissection itself do not impact on RNA quality. However, the overall process of the LCM procedure affects the RNA quality, resulting in a bias in the gene profiles. Nonetheless, this bias did not prevent accurate determination of a CLO-specific molecular signature. Thus, gene-profiling analysis of microdissected foci, identified by H&E staining may provide insight into the mechanisms underlying non-genotoxic hepatocarcinogenesis in the rat by allowing identification of specific genes that are regulated by CLO in early pre-neoplastic foci.

Animals↗

PubMed2001

Quantitative gene expression analysis in microdissected archival formalin-fixed and paraffin-embedded tumor tissue.

Formalin-fixed, paraffin-embedded tissue is the most widely available material for retrospective clinical studies. In combination with the potential of genomics, these tissues represent an invaluable resource for the elucidation of disease mechanisms and validation of differentially expressed genes as novel therapeutic targets or prognostic indicators. We describe here an approach that, in combination with laser-assisted microdissection allows quantitative gene expression analysis in formalin-fixed, paraffin-embedded archival tissue. Using an optimized RNA microscale extraction procedure in conjunction with real-time quantitative reverse transcriptase-polymerase chain reaction based on fluorogenic TaqMan methodology, we analyzed the expression of a panel of cancer-relevant genes, EGF-R, HER-2/neu, FGF-R4, p21/WAF1/Cip1, MDM2, and HPRT and PGK as controls. We demonstrate that expression level determinations from formalin-fixed, paraffin-embedded tissues are accurate and reproducible. Measurements were comparable to those obtained with matching fresh-frozen tissue and neither fixation grade nor time significantly affected the results. Laser microdissection studies with 5-microm thick sections and defined numbers of tumor cells demonstrated that reproducible quantitation of specific mRNAs can be achieved with only 50 cells. We applied our approach to HER-2/neu quantitative gene expression analysis in 54 microdissected tumor and nonneoplastic archival samples from patients with Barrett's esophageal adenocarcinoma and showed that the results matched those obtained in parallel by fluorescence in situ hybridization and immunohistochemistry. Thus, the combination of laser-assisted microdissection and real-time TaqMan reverse transcriptase-polymerase chain reaction opens new avenues for the investigation and clinical validation of gene expression changes in archival tissue specimens.

Animals↗

PubMed2002

cDNA array hybridization after laser-assisted microdissection from nonneoplastic tissue.

Differential gene expression can be investigated effectively by cDNA arrays. Because tissue homogenates result inevitably in an average expression of a bulk of different cells, we aimed to combine mRNA profiling with cell-type-specific microdissection. Using a polymerase chain reaction (PCR)-based preamplification technique, the expression profile was shown to be preserved. We modified the existing protocol enabling to apply the total amount of extracted RNA from microdissected cells. A mean amplification factor of nearly 1000 allowed to reduce the demand of initial RNA to approximately 10 ng. This technique was used to investigate intrapulmonary arteries from mouse lungs ( approximately 500 cell equivalents). Using filters with 1176 spots, three independent experiments showed a high consistency of expression for the preamplified cDNAs. These profiles differed primarily from those of total lung homogenates. Additionally, in experimental hypoxia-induced pulmonary hypertension, amplified cDNA from intrapulmonary vessels of these lungs was compared to cDNA from vessels dissected from normoxic lungs. Validation by an alternative method was obtained by linking microdissection with real-time polymerase chain reaction (PCR). As suggested by the array data, nine selected genes with different factors of up-regulation were fully confirmed by the PCR technique. Thus, a rapid protocol is presented combining microdissection and array profiling that demands low quantities of initial RNA to assess reliably cell-type-specific gene regulation even within nonneoplastic complex tissues.

Animals↗

PubMed2000

Sensitive immunoassay of tissue cell proteins procured by laser capture microdissection.

Coupling laser capture microdissection (LCM) with sensitive quantitative chemiluminescent immunoassays has broad applicability in the field of proteomics applied to normal, diseased, or genetically modified tissue. Quantitation of the number of prostate-specific antigen (PSA) molecules/cell was conducted on human prostate tissue cells procured by LCM from fixed and stained frozen sections. Under direct microscopic visualization, laser shots 30 microm in diameter captured specific cells from the heterogeneous tissue section onto a polymer transfer surface. The cellular macromolecules from the captured cells were solubilized in a microvolume of extraction buffer and directly assayed using an automated (1.5 hour) sandwich chemiluminescent immunoassay. Calibration of the chemiluminescent assay was conducted by developing a standard curve using known concentrations of PSA. After the sensitivity, precision, and linearity of the chemiluminescent assay was verified for known numbers of solubilized microdissected tissue cells, it was then possible to calculate the number of PSA molecules per microdissected tissue cell for case samples. In a study set of 20 cases, using 10 replicate samples of 100 laser shots per sample, the within-run (intraassay) SD was approximately 10% of the mean or less for all cases. In this series the number of PSA molecules per microdissected tissue cell ranged from 2 x 10(4) to 6. 3 x 10(6) in normal epithelium, prostate intraepithelial neoplasia (PIN), and invasive carcinoma. Immunohistochemical staining of human prostate for PSA was compared with the results of the soluble immunoassay for the same prostate tissue section. Independent qualitative scoring of anti-PSA immunohistochemical staining intensity paralleled the LCM quantitative immunoassay for each tissue subpopulation and verified the heterogeneity of PSA content between tissue subpopulations in the same case. Extraction buffers were successfully adapted for both secreted and membrane-bound proteins. This technology has broad applicability for the quantitation of protein molecules in pure populations of tissue cells.

Calibration↗

PubMed2000

Detection of gene amplification in archival breast cancer specimens by laser-assisted microdissection and quantitative real-time polymerase chain reaction.

Gene amplification is one of the most important mechanisms leading to deregulated gene expression in cancer. The exact quantitative detection of this frequent genomic alteration in solid tumors is often hampered by an admixture of nonneoplastic bystander and stroma cells. To overcome this obstacle and to develop an objective quantitative method we have combined laser-assisted microdissection of tumor cells with the novel 5'-exonuclease-based real-time polymerase chain reaction (PCR) assay. The latter method enables the highly reproducible exact quantification of minute amounts of nucleic acids. As a model system amplification of c-erbB2/Her-2/neu gene and the adjacent topoisomerase IIalpha gene was determined in paraffin-embedded breast cancer specimens (n = 23) after immunohistochemical labeling and laser-based microdissection of tumor cells. The high sensitivity of real-time PCR enabled the reliable and objective detection of low-level amplifications in as few as 50 cells from archival tissue sections. Low-level amplifications were shown to escape from detection unless tumor cells were isolated by microdissection. In selected cases intratumor heterogeneity was demonstrated using areas of approximately 50 to 100 cells. This novel approach combining immunohistochemistry, laser microdissection, and quantitative kinetic PCR allows morphology-guided studies in archival tissue specimens and will enable the exact quantification of gene copy numbers in even small and precancerous lesions.

Antigens, Neoplasm↗

PubMed1998

Analysis of mRNA from microdissected frozen tissue sections without RNA isolation.

Molecular study of gene expression in solid tumors is based largely on mRNA extracted from crushed frozen tumor samples. As most tumors are heterogeneous in composition, molecular alterations acquired by neoplastic cells may be masked by normal epithelial, stromal, and inflammatory cells, which may make up a significant volume of many tumors. We have developed a technique whereby reverse transcription polymerase chain reaction (RT-PCR) can be performed on lesions microdissected directly from frozen tumor sections. This allows for molecular analysis of mRNA from histologically homogeneous cell populations. Cryostat sections are placed onto a thin layer of 2% agarose on a glass slide and stained briefly. Microdissected tissue is immersed in a freezing solution to lyse the cells; aliquots are used directly in RT-PCR reactions without further purification. We successfully amplified cDNA fragments of the beta2-microglobulin, p21Waf1, and BRCA1 genes from small microdissected lesions. Also, we examined the effect of varying thickness of cryostat sections (20 versus 40 microm) and several tissue staining dyes. We estimate that a small microdissected region, containing no more than 200 cells, can provide enough mRNA to make cDNA for 80 to 100 PCR reactions. We believe that this technique will be a useful tool to study gene expression in histologically defined tissues.

BRCA1 Protein↗

PubMed1998

Loss of heterozygosity on chromosome 11p15 during histological progression in microdissected ductal carcinoma of the breast.

Microdissection of histologically identifiable components from formalin-fixed, paraffin-embedded tissue sections allows molecular genetic analyses to be correlated directly with pathological findings. In this study, we have characterized loss of heterozygosity (LOH) at chromosome 11p15 at different stages of progression in microdissected tumor components from 115 ductal carcinomas of the breast. Microdissected foci of intraductal, infiltrating, and metastatic tumors were analyzed to determine the stage of progression at which LOH at 11p15 occurs. LOH was detected in 43 (37%) of 115 cases. Foci of intraductal carcinoma could be microdissected from 85 cases, of which 30 (35%) showed LOH at some stage of progression. LOH was detected in the intraductal component in 26 of these 30 cases. Interstitial deletions were characterized by using a panel of 10 highly polymorphic markers. The smallest region of overlap (SRO) for LOH at 11p15 was bounded by the markers D11S4046 and D11S1758. LOH at 11p15.5 showed no correlation with estrogen receptor status, the presence of positive lymph nodes, tumor size, histological grade, or long-term survival. We conclude that 11p15 LOH usually occurs early in breast cancer development but less frequently does not develop until the infiltrating or metastatic stages of tumor progression.

Breast Neoplasms↗