Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Microdissection”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 235 records · Page 13Linked to original sources
PubMed2001

Small-sample total RNA purification: laser capture microdissection and cultured cell applications.

Gene expression studies require analysis of RNA, but isolation of total RNA from very small samples by traditional methods can be difficult and inefficient. The Absolutely RNA microprep kit provides a convenient method for isolating total RNA from small numbers of cells such as those harvested by laser capture microdissection (LCM). The protocol includes binding of RNA to a solid support, thus eliminating the need for organic extraction and alcohol precipitation. DNase digestion on the solid support reduces or eliminates DNA contamination and minimizes RNA handling. Efficient washing removes contaminants, and elution in a small volume of buffer results in high-purity RNA at a concentration appropriate for demanding applications such as RT-PCR. RNA isolated from as few as 200 laser capture microdissected brain tumor cells resulted in detection of low, medium, and highly expressed genes by conventional and real-time RT-PCR.

3T3 Cells↗
PubMed1999

Laser capture microdissection of single cells from complex tissues.

Laser capture microdissection (LCM) is a new method used to select and procure cell clusters from tissue sections. Once captured, the DNA, RNA or protein can be easily extracted from the isolated cells and analyzed by conventional PCR, reverse transcription (RT)-PCR or polyacrylamide gel electrophoresis, including protein zymography for specific macromolecular changes. In LCM, a thermoplastic polymer coating [ethylene vinyl acetate (EVA)] attached to a rigid support is placed in contact with a tissue section. The EVA polymer over microscopically selected cell clusters is precisely activated by a near-infrared laser pulse and then bonds to the targeted area. Removal of the EVA and its support from the tissue section procures the selected cell aggregates for molecular analysis. This initial NIH LCM approach using a flat transfer EVA film has been recently commercialized and has proven to be an effective routine microdissection technique for subsequent macromolecular analysis in many laboratories around the world. However, reliable and precise capture of individual cells from tissue sections has been difficult to perform with the current LCM instruments. In this report, we describe the capture of individual cells with a new NIH LCM microscope, which epi-irradiates the EVA polymer overlying individual cells with 1-ms laser pulses focused to 6 microns. A computer-controlled arm precisely positions a 40-micron-wide strip of a cylindrical EVA surface onto a sample with a light contact force (ca. 0.1 g). The small contact force and contact area on the film on the sample diminishes nonspecific transfer to negligible levels. By slightly rotating the cylinder to provide a renewable transfer surface, concentration of a distinct cell type on a single cylinder is possible. Using this novel adaptation, we demonstrate the rapid and practical capture of single cells from different types of tissue sections, including immunostained cells.

Animals↗
PubMed1999

Directly inherited partial trisomy of chromosome 6p identified in a father and daughter by chromosome microdissection.

Cytogenetic analysis of a 4 year old girl with developmental delay and dysmorphic features showed extra chromosomal material of unknown origin on 20p (46,XX,add(20)(p13)). Familial chromosome studies showed direct inheritance of add(20)(p13) from the father, who had a similar, albeit milder, phenotype. Fibroblast chromosome studies of the father showed no karyotype mosaicism. The additional material could not be identified on the basis of the G banding pattern owing to its small size and ambiguous banding pattern. Chromosome microdissection of the unknown material was performed, the DNA was amplified and labelled using degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR) and reverse painted to the proband's cells to show the karyotype 46,XX,der(20)t(6;20) (p23;p13), conferring partial trisomy 6p and presumed partial monosomy for 20p. Chromosome microdissection has made possible the first reported case of directly inherited partial trisomy 6p.

Child, Preschool↗
PubMed1999

Utilization of microdissection and the polymerase chain reaction for the diagnosis of adrenal cortical carcinoma in fine-needle aspiration cytology.

BACKGROUND: Loss of heterozygosity (LOH) for several tumor suppressor genes (including loci on 3p, 1p, and 17p,) has been documented in surgical specimens of adrenal cortical carcinomas (ACCA) without accompanying losses in benign hyperplastic and adenomatous adrenal cortical lesions (ACL). This disparate pattern of LOH raises the possibility of exploitation of these differences for diagnostic utilization. Cytologic differentiation of benign versus malignant ACL may be impossible based solely on fine-needle aspiration (FNA) material. The authors attempted to extrapolate the genetic findings on surgical specimens to FNA specimens of ACL to determine whether LOH studies could be utilized as a definitive diagnostic tool. METHODS: Microdissection of archival material was performed on FNAs of ten ACCAs (stained with the Papanicolaou and Diff-Quik stains) with corresponding histologic material (stained with hematoxylin and eosin), one FNA of a benign ACL, and three touch preparations of benign adrenal cortex. LOH analysis was performed by polymerase chain reaction (PCR) with flanking markers for the following putative tumor suppressor genes: p53 (17p13; TP53), 1p (1p36; D1S165), and the von Hippel-Lindau gene at 3p25 (D3S1038 and D3S1110). RESULTS: Similar results were obtained with cytologic and histologic material. As expected, benign ACL showed no LOH for the markers examined. Of the informative ACCA cases, 70% showed LOH for at least 1 of the 3 markers tested on both FNA and histologic samples. For all cases with amplifiable DNA, there was a 100% concordance rate for LOH between cytologic and histologic material, with at least 7 of the 10 cytologic samples originating from metastatic lesions and all of the surgical material originating from the primary adrenal neoplasm. CONCLUSIONS: The results of this study suggest that the combination of microdissection and PCR for LOH of p53, 1p, and 3p25 from FNA material has the potential to be utilized to distinguish ACCA from benign ACL in informative cases. It also shows a 100% concordance rate between metastatic and primary ACCAs for the losses observed, a finding that can be extremely useful for the definitive identification of metastatic lesions. Archival cytologic preparations of ACCA are a reliable source of DNA for LOH studies. [See editorial counterpoint on pages 173-5 and reply to counterpoint on pages 176-7, this issue.] Cancer (Cancer Cytopathol)

Adrenal Cortex Neoplasms↗
PubMed1999

[Laser-assisted microdissection and molecular analysis at the cellular level].

Developments of molecular biological techniques have allowed the analysis of small tissue samples. In order to obtain optimal results it is essential to work with pure cell populations. The procurement of pure samples has, however, been one of the main limiting factors in biomedical research. Recently developed systems for laser-assisted microdissection now allow the isolation of pure cell populations from tissue sections, even at a single cell level, that are suitable for subsequent molecular analyses. Since morphological features can be directly related to molecular characteristics, studies concerning molecular genetic backgrounds underlying disease can be performed more easily and accurately. By future combined use of laser microdissection and new molecular analysis methods, the applications for laser-micro-dissection will increase further.

Cell Separation↗
PubMed2000

Quantitative gene expression analysis in microdissected archival tissue by real-time RT-PCR.

Laser-assisted microdissection is a powerful tool for the analysis of morphologically defined cell populations. We report here that the combination of microdissection and real-time RT-PCR technologies together with an optimized RNA microscale extraction procedure allows the reproducible determination of gene expression levels in archival formalin-fixed, paraffin-embedded tissue samples.

Dissection↗
PubMed2000

PCR artifacts in LOH and MSI analysis of microdissected tumor cells.

Polymerase chain reaction (PCR) analysis to study loss of heterozygosity (LOH) and microsatellite instability (MSI) in tumors is widely used. Microdissection techniques are applied to obtain tumor-specific tissue cells. By microdissection, however, the amount of template DNA extracted may vary considerably and interfere with optimal PCR amplification. To circumvent LOH and MSI misinterpretations due to low DNA input, we have assessed the critical level of DNA input for reliable PCR analysis. PCR analysis was performed by using 18 polymorphic markers (mono-, di-, tri-, and tetranucleotide) on DNA derived from both paraffin-embedded, formalin-fixed, and fresh frozen tumor specimens at template input levels ranging from 0.05 to 25.0 ng. We show a highly significant relation between DNA input and the occurrence of LOH and MSI artifacts. Furthermore, for DNA extracted from paraffin-embedded material, the percentage of LOH artifacts is significantly higher compared with DNA extracted from frozen tissue. For reliable PCR analyses using a mono-, di-, tri-, or tetranucleotide marker, a minimum of 10.0 ng DNA is required when DNA is isolated from formalin-fixed, paraffin-embedded tissue and 5.0 ng when isolated from fresh frozen tissue. HUM PATHOL 31:1414-1419.

Alleles↗
PubMed2001

The detection of p53 gene mutation using a microdissection technique in primary intracranial germ cell tumors.

Using a microdissection technique, the contribution of the p53 mutation to tumorigenesis and prognosis in each histological subtype of the intracranial germ cell tumors (GCTs) was evaluated. Nineteen patients had primary intracranial GCTs, including 4 germinomas (GEs), 4 teratomas (TEs), 1 mixed tumor of GE and TE, and 10 mixed GCTs containing non-germinomatous malignant germ cell tumors (NG-MGCTs). After microdissection of specific subtypes, genomic DNA was screened for mutations in exons 5-8 of the p53 gene, using the dideoxyfingerprinting (ddF) followed by direct DNA sequencing. The direct sequencing revealed a total of six mutations in PCR products derived from the five cases (26%) which showed mobility shifts in ddF. Among the six mutations detected, four were missense mutations and two were silent. Missense mutations of the p53 gene tended to occur more frequently in the NG-MGCT component than in the GE or TE components (3/15 vs. 1/12 vs. 0/13). The incidence of missense mutations was not different between the survivors (3/13) and the deceased (1/6). This study suggests the possible role of the p53 gene in the tumori-genesis of NG-MGCT. However, p53 gene mutation did not correlate with the prognosis of NG-MGCT.

Adult↗
PubMed1996

Acute effects of morphine on Substance P concentrations in microdissected regions of guinea-pig brain.

The present study investigated the effects of acute morphine treatment and of naloxone-induced morphine withdrawal on Substance P (SP) concentrations in microdissected regions of the guinea-pig brain. Guinea-pigs, which were treated with a single dose of morphine sulphate (15mg/kg s.c.), received naloxone hydrochloride (15mg/kg s.c.) after 2h. Control animals received injections of saline, saline and naloxone, or morphine and saline. Locomotor and behavioural activities were measured throughout the experiments. Animals were killed 0.5h after naloxone administration, brains were removed and SP-like immunoreactivity (SP-LI) was measured in microdissected regions using radioimmunoassay. Morphine significantly increased the concentration of SP-LI in the central nucleus of the amygdala, but reduced SP-LI overall in the mesencephalon. Guinea-pigs pretreated with morphine and then given naloxone to precipitate withdrawal showed no change in SP-LI concentrations in any brain region, compared with control animals, despite an increase in locomotor activity and other behaviours characteristic of opioid withdrawal in guinea-pigs.

Journal Article↗
PubMedDate not supplied

The role of tissue microdissection in cancer research.

Tissue microdissection is a laboratory method that is used to procure specific cells or cell populations from a histology slide under direct microscopic visualization. The recovered cells can be studied with a variety of DNA, messenger RNA, and protein analysis methods, including new high-throughput gene expression and proteomics technologies. This approach is permitting investigators to comprehensivelyexamine the molecular anatomy of cells in tissue sections forthe first time. This article reviews the development and evolution of tissue microdissection techniques, summarizes examples of research studies, and discusses related challenges that the research community must address. Additional information and complete laboratory protocols are available on a website at http://cgap-mf.nih.gov/.

DNA, Neoplasm↗
PubMed2001

Identification and validation of P311 as a glioblastoma invasion gene using laser capture microdissection.

The mRNA expression profiles from glioblastoma cells residing at the tumor core and invasive rim of a human tumor resection were compared. From a single tumor specimen, 20,000 single cells from each region were collected by laser capture microdissection. Differential expression of 50-60 cDNA bands was detected. One of the sequences overexpressed by the invasive cells showed 99% homology to the P311 gene, the protein product of which is reported to localize at focal adhesions. Relative overexpression of P311 by invading glioblastoma cells compared with tumor core was confirmed by quantitative reverse transcription-PCR of six glioblastoma specimens after laser capture microdissection collection of rim and core cells. In vitro studies using antisense oligodeoxynucleotides and integrin activation confirmed the role of P311 in supporting migration of malignant glioma cells. Immunochemistry studies confirmed the presence of the P311 protein in tumor cells, particularly at the invasive edge of human glioblastoma specimens.

Amino Acid Sequence↗
PubMed1992

Frequency and significance of cervicomediastinal lymph node metastases in medullary thyroid carcinoma: results of a compartment-oriented microdissection method.

The frequency and significance of cervicomediastinal lymph node metastases have been investigated in 82 medullary thyroid carcinoma (MTC) patients retrospectively comparing two surgical techniques of lymph node dissection: selective lymphadenectomy (n = 63) versus compartment-oriented microdissection (n = 35). No positive correlation was observed between primary tumor size and the number of lymph node metastases. In patients with lymph node metastases proven histologically, 42% showed only cervical involvement (35% unilateral--type A, 7% bilateral--type B) and 22% cervicomediastinal lymph node involvement (15% cervico-unilateral and mediastinal--type C, 7% cervicobilateral and mediastinal--type D). Biochemical cure was 83% in node-negative patients but only 21% in node-positive patients. In node-positive MTC, calcitonin normalization was achieved in none with bilateral lymph node involvement but only in those unilateral lymph node metastases (31% in type A, 17% in type C). Survival and biochemical cure are significantly improved by application of the compartment-oriented microdissection method more so at primary surgery than at reoperation.

Carcinoma↗
PubMed1991

[Technique for the microdissection of human high resolution chromosome subsequent polymerase chain reaction and microcloning].

In this paper is reported a simple technique for the microdissection of specific regions of human high resolution chromosome subsequent PCR and microcloning. This technique was successfully used to microdissect 4 chromosomal pieces from the distal one third from band 11.2 to the terminal of the short arm of Y chromosome where the testis determining factor located, PCR 30 cycles, and obtained 3.6 x 10(4) clones. This technique the highlight to establish a gene library of many specific regions of human chromosome hereafter.

Cloning, Molecular↗
PubMed1991

Microdissection of human high resolution banded chromosome, polymerase chain reaction and microcloning.

A simple technique for microdissection of specific region of human high resolution banded chromosome, followed by polymerase chain reaction (PCR), and microcloning was successfully used to microdissect 4 chromosomal pieces from the distal one third from band 11.2 to the terminal of the short arm of Y chromosome where the testis determining factor is located; 3.6 x 10(4) clones were obtained after 30 cycles of PCR. We analysed 41 clones with insert. The size of insert ranges from 140 to 350 bp (average 250 bp). A Southern blot analysis was done for one of them, and a 2.5 kb Hind III fragment was detected.

Cloning, Molecular↗
PubMed1988

The gill arch of the striped bass, Morone saxatilis. III. Morphology of the basal lamina as revealed by various ultrasonic microdissection procedures.

The ultrastructure of the surface epithelium and the associated basal lamina of the gill arches of the striped bass, Morone saxatilis, were investigated with the scanning electron microscope following complete or partial removal of the epithelium by ultrasonic microdissection. The microdissection procedures employed various combinations of the techniques of aldehyde fixation, treatment with borate, and extensive osmication followed by mild sonication. Generally, aldehyde fixation increases intercellular adhesion, excess osmication increases tissues brittleness, and borate treatment causes extensive tissue dissociation. However, the degree of epithelial removal following sonication of tissues treated with these various procedures varies considerably with specimen structure, shape, proximity to adjacent structures and freedom to vibrate during sonication. The basal lamina exhibits a smooth contour over most of the gill surface with the exception of the short gill rakers where it formed cones within the taste bud cores, and on the respiratory lamellae where it closely mimicked the underlying capillary network.

Animals↗
PubMed1986

Ultrasonic microdissection of immature intermediate human placental villi as studied by scanning electron microscopy.

The human placenta during the first 20 weeks of gestation undergoes rapid and extensive morphological changes. Near the end of this period, the most predominant type of villus present is the immature intermediate placental villus. In order to visualize this complex structure with scanning electron microscopy (SEM), we have developed a microdissection technique to expose tissue components of the placental villus while retaining its normal histological architecture. Placental villi were initially fixed in Karnovsky's fixative, buffered formalin, or 2% osmium tetroxide solution prior to exposure to connective tissue enzymes or detergents alone or in combination. Samples were dehydrated through 100% acetone and ultrasonicated at 80 kHz for 15 minutes prior to critical point drying and SEM examination. The most satisfactory microdissections were obtained by using a combined detergent/ultrasonication technique. By means of this procedure it was possible to remove the syncytiotrophoblast to expose the underlying cytotrophoblast, basal lamina and the stromal core components of the villi. The selective removal of these structures revealed the 3-dimensional relationships of the stromal channels, reticulum cells and Hofbauer cells. Of interest was the pattern of fetal capillaries coursing parallel to the long axis of each villus and terminating in a vascular knot at the tip.

Cell Fractionation↗
PubMed1978

Cystic kidneys, renal dysplasia and microdissection data in 5 children with congenital valvular urethral obstruction.

A survey of the findings in 5 children with urethral obstruction due to valves is presented. The microdissection data found in the 7 kidneys of this series were compared. The microdissection findings in all 7 kidneys are mostly the same with a continuum of processes ranging from relatively mild to severe derangements. We could not find two varieties of renal change designated type II or IV by potter. The characterization of the lesions by nephron dissection always approached those of the type II (multicystic, dysplastic kidney). Renal dysplasia was associated with extrarenal malformations in 2 of our cases and we therefore believe that the significance of the dysplasia lies rather in the period of the insult during organogenesis than in the obstruction.

Autopsy↗
PubMed1978

Structural changes of experimental glomerulonephritis in rats as revealed by microdissection.

Microdissection revealed striking alterations in the nephrons of animal models AICN and anti-GBMN. The most severely damaged AICN kidney in the series presented marked heterogeneity in the size and deformity of the proximal tubules, corresponding to the diverse and variegated nephrons described by Oliver in chronic Bright's disease. The severely damaged anti-GBMN kidneys revealed widespread alterations in the proximal tubules, which, however, tended to be fairly uniform among the affected nephrons. This uniformity, perhaps, reflected the shorter duration of disease in the anti-GBMN animals. The most characteristic proximal tubular alteration in either the AICN or anti-GBMN animals was that combining atrophy of the pars convoluta and hypertrophy of the pars recta in same tubule. The largest and smallest nephrons encountered in the entire study were found in the most severly damaged kidney in the AICN. This finding reflected the simultaneous existence of regressive and progressive changes in this kidney, as certain nephrons underwent hypertrophy to compensate for the atrophy and disappearance of others. The remarkable functional glomerulotubular balance of single nephrons known to exist in both AICN and anti-GBMN was matched by the evidence presented here of structural glomerulotubular balance among the severely altered nephrons of a representative kidney from each group. Microdissection studies directed toward the characterization of the nephronic alterations in both membranous glomerulonephritis and proliferative glomerulonephritis of man should be of interest in relation to the findings in these experimental rat models of human disease.

Animals↗