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 487 records · Page 27Linked to original sources
PubMed1998

Rat estrogen receptor-alpha and -beta, and progesterone receptor mRNA expression in various prostatic lobes and microdissected normal and dysplastic epithelial tissues of the Noble rats.

Semiquantitative RT-PCR was used to determine if transcripts of the two estrogen receptor (ER) subtypes, ER alpha and ER beta, and the progesterone receptor (PR) are differentially expressed and/or regulated in the various normal lobes of the Noble (NBL) rat prostate. We found that ER beta mRNA was present at comparable, high levels in all three major prostatic lobes: dorsal (DP), lateral (LP) and ventral (VP) prostate. ER alpha mRNA was, however, expressed at low levels among the various lobes in the following descending order of abundance: LP>DP>VP. Expression of PR transcript was low and paralleled the expression pattern of ER alpha mRNA. Treatments of rats with testosterone (T) plus estradiol-17beta (E2) (T+E2) or T alone induced no discernible alterations in ER alpha, ER beta, and PR mRNA levels in the VP, DP and LP, while those with E2 caused a general decline in the expression of all three transcripts. We then studied the expression of the three receptors in the normal and dysplastic epithelium of the dorsolateral prostates (DLPs) of rats treated with T+E2. Comparable levels of ER beta mRNA were found in microdissected dysplastic and normal epithelia. In contrast, significantly higher levels of PR mRNA were present in epithelial samples from dysplastic acini. ER alpha mRNA was not detected in any of the microdissected epithelial samples. Results from this study suggest that upregulation of PR mRNA expression, likely mediated via ER beta action, is involved in the genesis of T+E2-induced dysplasia in this animal model.

Animals↗
PubMed2003

Mutational analysis of the androgen receptor using laser capture microdissection and direct sequencing.

Molecular analysis of prostate cancer specimens is complicated by tumor heterogeneity and admixture of malignant cells with benign epithelium, stroma and inflammatory cells. Laser Capture Microdissection allows procurement of selected groups of cells from specific areas of tissue sections. We have used Laser Capture Microdissection to obtain pure samples of malignant prostate epithelial cells from frozen radical prostatectomy specimens. Methods for DNA extraction, androgen receptor amplification and exon sequencing have been optimized. These methods should be adaptable for molecular analysis of DNA, RNA and protein from other complex tissues.

DNA Mutational Analysis↗
PubMed2002

Gene expression profiling using laser capture microdissection.

Human disease is governed by a complex array of cellular populations and sub-population. Gene expression profiling is proving an important means of understanding and classifying pathophysiologic processes by identifying genes, gene pathways and pathway networks not previously known to be associated with particular diseases. However, disease-associated gene expression can be obscured by surrounding 'normal' tissue. Laser capture microdissection allows gene expression analysis of pooled single cells, cell subpopulations and cell populations. Analysis of laser capture microdissection-procured cells will allow a better understanding of the cellular components of disease.

DNA, Complementary↗
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↗
PubMed2002

High-quality RNA from cells isolated by laser capture microdissection.

Laser capture microdissection (LCM) provides a rapid and simple method for procuring homogeneous populations of cells. However, reproducible isolation of intact RNAfrom these cells can be problematic; the sample may deteriorate before or during sectioning, RNA may degrade during slide staining and LCM, and inadequate extraction and isolation methods may lead to poor recovery. Our report describes an optimized protocol for preparation of frozen sections for LCM using the HistoGene Frozen Section Staining Kit. This slide preparation method is combined with the PicoPure RNA Isolation Kitfor extraction and isolation of RNA from low numbers of microdissected cells. The procedure is easy to perform, rapid, and reproducible. Our results show that the RNA isolated from the LCM samples prepared according to our protocol is of high quality. The RNA maintains its integrity as shown by RT-PCR detection of genes of different abundance levels and by electrophoretic analysis of ribosomal RNA. RNA obtained by this method has also been used to synthesize probes for interrogating cDNA microarray analyses to study expression levels of thousands of genes from LCM samples.

Animals↗
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↗
PubMed2006

RCL2, a new fixative, preserves morphology and nucleic acid integrity in paraffin-embedded breast carcinoma and microdissected breast tumor cells.

Methacarn and RCL2, a new noncrosslinking fixative, were compared to formalin-fixed or frozen tissue samples of the same invasive breast carcinoma and were evaluated for their effects on tissue morphology and immunohistochemistry as well as DNA and RNA integrity. The histomorphology of methacarn- or RCL2-fixed paraffin-embedded tumors was similar to that observed with the matched formalin-fixed tissues. Immunohistochemistry using various antibodies showed comparable results with either fixative, leading to accurate breast tumor diagnosis and determination of estrogen and progesterone receptors, and HER2 status. Methacarn and RCL2 fixation preserved DNA integrity as demonstrated by successful amplification and sequencing of large DNA amplicons. Similarly, high-quality RNA could be extracted from methacarn- or RCL2-fixed paraffin-embedded MCF-7 cells, whole breast tumor tissues, or microdissected breast tumor cells, as assessed by electropherogram profiles and real-time reverse transcriptase-polymerase chain reaction quantification of various genes. Moreover, tissue morphology and RNA integrity were preserved after 8 months of storage. Altogether, these results indicate that methacarn, as previously shown, and RCL2, a promising new fixative, have great potential for performing both morphological and molecular analyses on the same fixed tissue sample, even after laser-capture microdissection, and can open new doors for investigating small target lesions such as premalignant breast lesions.

Breast Neoplasms↗
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↗
PubMed2002

Analysis of microdissected prostate tissue with ProteinChip arrays--a way to new insights into carcinogenesis and to diagnostic tools.

Prostate carcinomas are one of the most common malignancies in western societies. The pathogenesis of this tumor is still poorly understood. These tumors present with two characteristic features: epithelial-mesenchymal interactions, which play a pivotal role for tumor development and most of clinically manifest cancers arise in prostate proper compared to a minority of tumors which develop in the transitional zone. Deciphering the epithelial-mesenchymal cross talk and identification of molecular pecularities of the sub-populations of cells in different zones can therefore help understanding carcinogenesis and development of new, non-invasive tools for the diagnosis and prognosis of prostate carcinomas which has remained a challenge until today. A ProteinChip array technology (SELDI = surface enhanced laser desorption ionization) has been developed recently by Ciphergen Biosystems enabling analysis and profiling of complex protein mixtures from a few cells. This study describes the analysis of approximately 500-1000 freshly obtained prostate cells by SELDI-TOF-MS (surface enhanced laser desorption ionization time-of-flight mass spectrometry). Pure cell populations of stroma, epithelium and tumor cells were selected by laser assisted microdissection. Multiple specific protein patterns were reproducibly detected in the range from 1.5 to 30 kDa in 28 sub-populations of 4 tumorous prostates and 1 control. A specific 4.3 kDa peak was increased in the prostate tumor stroma compared to normal prostate proper and transitional zone stroma and increased in prostate tumor glands compared to normal prostate proper and transitional zone glands. Coupling laser assisted microdissection with SELDI provides tremendous opportunities to identify cell and tumor specific proteins to understand molecular events underlying prostate carcinoma development. It underlines the vast potential of this technology to better understand pathogenesis and identify potential candidates for new specific biomarkers in general which could help to screen for and distinguish disease entities, i.e. between clinically significant and insignificant carcinomas of the prostate.

Aged↗
PubMed2002

Identification of cyclin D1 mRNA overexpression in B-cell neoplasias by real-time reverse transcription-PCR of microdissected paraffin sections.

PURPOSE: Overexpression of cyclin D1 mRNA and protein as a result of the chromosomal translocation t(11;14)(q13;q32) is a highly specific molecular marker of mantle cell lymphoma, but cyclin D1 dysregulation can also be found in other B-cell neoplasias. The aim of the study was to develop a precise and reliable tool for quantitation of cyclin D1 mRNA suitable for archival clinical specimens. EXPERIMENTAL DESIGN: A real-time reverse transcription-PCR (RT-PCR) assay was used to quantitate cyclin D1 mRNA copy numbers. Using 2000 microdissected cells as template, 104 formalin-fixed, paraffin-embedded lymph node, spleen, and decalcified bone marrow biopsies from a panel of 95 cases of B-cell non-Hodgkin's lymphomas (B-NHLs) were analyzed. In addition, cyclin D1 protein expression was assessed by immunohistochemistry. RESULTS: Strong cyclin D1 mRNA overexpression was detected in mantle cell lymphomas (23 of 23), hairy cell leukemias (5 of 19), and multiple myelomas (7 of 23) with particularly high levels in 2 of the latter cases. Intermediate transcript levels were found in 5 of 23 multiple myelomas and 7 of 19 hairy cell leukemias. B-cell chronic lymphocytic leukemias (10 of 10), follicular lymphomas (9 of 9), mucosa-associated lymphoid tissue lymphomas (5 of 5) and reactive lymphoid tissues with the exception of normal spleen had no or very low cyclin D1 expression. In comparison with real-time RT-PCR, immunohistochemistry showed a lower level of sensitivity, more variability, and did not allow accurate quantitation. CONCLUSIONS: Real-time RT-PCR for cyclin D1 mRNA is an excellent tool for the differential diagnosis of B-NHLs and, in combination with microdissection, a powerful approach for retrospective trials using archival clinical specimens as tissue source. Furthermore, real-time RT-PCR may help to identify subgroups of B-NHLs according to cyclin D1 mRNA copy numbers and to investigate the possible influence of different chromosomal breakpoints on cyclin D1 expression.

Chromosomes, Human, Pair 11↗