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PubMed1998

Microdissection-Based p53 Genotyping: Concepts for Molecular Testing.

p53 is the most commonly altered tumor-suppressor gene in humans, involved in the development and progression of many diverse forms of human cancer. Although much remains to be learned about the biology of this important growth regulatory gene, sufficient experience has been accumulated with respect to the occurrence and pattern of p53 mutational change to justify molecular diagnostic testing for specific objectives. The authors outline specific concepts for testing with particular emphasis for solid tumor molecular diagnostics. This article focuses on microdissection-based fixed tissue molecular analysis, introducing new considerations related to quality control appropriate for this type of methodology. Given the rapidly evolving nature of molecular genetics, the suggestions provided here should be viewed as flexible. Nevertheless, the concepts are intended to serve as a model for other oncogene/tumor suppressor-gene assays to be developed with the overall purpose of establishing informative integrated histopathologic/genetic molecular diagnostic testing to complement standard microscopic analysis.

Journal Article↗
PubMed1994

Lack of effect of atrial natriuretic peptide and urodilatin on vasopressin-stimulated cyclic AMP production in microdissected rat inner medullary collecting ducts.

It is unclear whether the diuretic effects of atrial natriuretic peptide (ANP) result, in part, from an inhibition of the renal actions of vasopressin. Moreover, accruing evidence suggests that the kidneys themselves may produce an ANP-like peptide, urodilatin, which shares many of the renal actions of ANP. The mechanism underlying the diuretic action of urodilatin has not yet been examined. Accordingly, we have investigated the potential modulatory actions of both ANP and urodilatin on vasopressin-stimulated cyclic AMP (cAMP) production in microdissected inner medullary collecting duct (IMCD) segments of rat kidney. ANP and urodilatin alone (at 10(-8) or 10(-6) M) had no demonstrable effect on cAMP accumulation in IMCD segments. Moreover, neither ANP nor urodilatin (each at 10(-6) M) significantly altered either the profile or the absolute magnitude of the cAMP response stimulated by vasopressin. These findings indicate that neither ANP nor urodilatin interacts with the vasopressin-sensitive adenylate cyclase site in the rat IMCD to contribute to its diuretic actions.

Adenylyl Cyclases↗
PubMed2003

Comparison of the effect of oestradiol, tamoxifen and raloxifene on nerve growth factor-alpha expression in specific neonatal mouse uterine cell types using laser capture microdissection.

Oral dosing of CD-1 mice on days 2-5 after birth with tamoxifen but not raloxifene disrupts the development of the myometrium, resulting in adult uterine adenomyosis. Using laser capture microdissection and RT-PCR we have investigated nerve growth factor (NGF) and cognate receptor expression in uterine cells of 6-day-old pups that may be important in early developmental changes that give rise to adenomyosis. NGF down-regulation is known to occur during terminal myogenic differentiation. NGF was found exclusively in endometrial luminal epithelium of controls. It was up-regulated 18-fold in the luminal epithelium following dosing with tamoxifen but not raloxifene. Western blotting for NGF protein in the whole uterus showed a 25-fold increase after tamoxifen treatment. Expression of the low affinity p75 neutrophin receptor (p75(NTR)) was twofold higher in the myometrium compared with luminal epithelium or stroma. This was not altered following tamoxifen treatment. There was no detectable expression of high affinity tyrosine kinase receptor (trkA(NGFR)). This study shows luminal epithelial cells of the endometrium primarily form NGF. This suggests that NGF normally regulates the differentiation of the mesenchyme into uterine myocytes through paracrine mechanisms and that an early disturbance of this process plays a key role in the subsequent development of adenomyosis.

Animals↗
PubMed2000

Comparison of two PCR techniques used in amplification of microdissected plant chromosomes from rice and wheat.

Linker adaptor-mediated PCR (LAM-PCR) and degenerate oligonucleotide primed PCR (DOP-PCR) are major ways to generate chromosome-specific libraries. In this study, these two PCR techniques were used to amplify the microdissected mitotic and meiotic chromosomes from rice and wheat. The results of amplifications were compared, and the advantages and limitations between the two techniques are presented.

Blotting, Southern↗
PubMed2000

Laser capture microdissection-generated target sample for high-density oligonucleotide array hybridization.

Current advances in biomolecular technology allow precise genetic fingerprinting of specific cells responsible for the pathogenesis of human diseases. This study demonstrates the feasibility of generating target samples from laser capture microdissection (LCM) tissues suitable for hybridization of high-density oligonucleotide arrays for gene expression profiling. RNA was successfully isolated by LCM from three paired specimens of oral cancer and linearly amplified using T7 RNA polymerase. Evaluation of the cDNA revealed that five of five cellular maintenance transcripts are detected. Biotinylated cRNA was generated and hybridized to the human Test 1 GeneChip probe arrays, which demonstrated that the RNA is of sufficient quality and integrity to warrant further analysis. Subsequent hybridization of the samples to the HuGenFL GeneChip probe arrays revealed that 26.5%-33.0% of the approximately 7000 represented genes are expressed in each of the six samples. These results demonstrate that LCM-generated tissues can generate sufficient quality cRNA for high-density oligonucleotide microarray analysis, an important step in determining comprehensive gene expression profiling using this high-throughput technology.

Biotinylation↗
PubMed2002

Identification of nitric oxide synthase neurons for laser capture microdissection and mRNA quantification.

An immunohistochemical technique was developed to visualize nitric oxide synthase (NOS)-immunopositive neurons in fresh-frozen tissue sections of rat brain for laser capture microdissection (LCM) and mRNA analysis. The effect of tissue fixation and the choice of fluorophore were investigated. Here we describe a rapid immunofluorescence protocol that allows the processing of fresh-frozen tissue sections within eight minutes and subsequent mRNA extraction and real-time PCR from pools of 20 NOS-immunopositive LCM neurons. The cellular complement of a subset of ionotropic glutamate receptors, specifically N-methyl-D-aspartate receptor subunit mRNAs, was examined because these receptor complexes are thought to mediate the effects of fast and slow glutamate excitotoxicity. Real-time PCR data revealed that striatal NOS interneurons express the mRNAs encoding NR1, NR2A, NR2B, and NR2D but not NR2C. These LCM mRNA data are consistent with previous in situ hybridization studies and demonstrate the utility of rapid immuno-LCM with real-time quantitative PCR for the study of mRNA abundance in discrete populations of neurons within the mammalian brain.

Animals↗
PubMed2003

Selection of chloroplasts by laser microbeam microdissection for single-chloroplast PCR.

Laser microbeam microdissection and laser pressure catapulting offer the possibility of separating cell compartments, thus allowing for contamination-free analysis. Using these methods, we were able to select single chloroplasts of Nicotiana tabacum. Starting from homogenized leaf material, chloroplasts were purified by differential centrifugation and applied directly onto a poly-ethylene-naphthalate membrane that was mounted on a microscope slide. Single chloroplasts were dissected under microscopic control and catapulted into a PCR tube. Subsequent PCR of a spacer region between the trnT and trnF genes verified the successful amplification of DNA from a single chloroplast. The advantage of this method compared to the use of capillaries or optical tweezers is that one is able to prepare high numbers of samples in a short time.

Base Sequence↗
PubMed1996

Analysis of loss of heterozygosity in microdissected tumor cells from cervical carcinoma using fluorescent dUTP labeling of PCR products.

The use of fluorescently labeled nucleotides (F-dUTP) in the PCR for genetic typing of microsatellite polymorphisms was investigated. Microdissected tumor cells from cervical squamous cancer biopsies were compared with cells from surrounding normal tissue for loss of heterozygosity (LOH) at the mismatch repair gene hMLH1. Removal of unincorporated F-dUTP before analysis is necessary to reduce background fluorescence; ethanol-precipitation was found to be as efficient as the use of a spin column for this purpose. The gel resolution was sufficient to distinguish alleles differing by about four nucleotides. Alleles differing by only one dinucleotide repeat were possible to identify, but the ratio of alleles was difficult to assert with any reliability due to the wide peaks obtained. Single primer-pair and nested amplification systems were shown in reconstitution experiments to reliably quantitate the ratio of alleles. Of 36 cervical cancer biopsies, amplification and typing of the hMLH1 microsatellite marker was successful in 20 cases. Among 9 informative (heterozygous) biopsies, 2 (22%) were found to show LOH.

Alleles↗
PubMed1998

Comparison of histologic stains for use in PCR analysis of microdissected, paraffin-embedded tissues.

We evaluated the effect of six different histologic stains on the productivity of PCR amplification of DNA isolated from paraffin-embedded tissue samples. The tissue was collected from glass slides by microdissection techniques, whereby tiny portions of tissue are visually identified through a microscope and selectively resected for subsequent DNA extraction and PCR amplification. We found that the success of PCR amplification depended on the type of histologic stain that was used to facilitate microscopic visualization of the undeparaffinized tissue section. The best results were obtained with methyl green and nuclear fast red, while Wright's stain yielded less PCR product. Two other stains, Evans blue and light-green SF yellowish (also known as the counterstain for geomori methenamine silver stain) yielded sufficient PCR products; however, their staining characteristics did not afford satisfactory visualization of nuclear chromatin to discriminate between benign and malignant cells. Our most significant finding was that a commonly used histologic stain, hematoxylin, failed to produce DNA templates that could be consistently amplified by PCR. In conclusion, it is prudent to avoid hematoxylin stains when preparing tissues as starting material for PCR. Among the remaining five stains that were evaluated, the best choice depends on the differential staining characteristics of the cells to be dissected.

Coloring Agents↗
PubMed1999

Laser microdissection and laser pressure catapulting for the generation of chromosome-specific paint probes.

Chromosome-specific paint probes provide a powerful tool with wide applications in cytogenetic analysis. Here, we present a new approach using UV-laser microbeam microdissection in combination with laser-pressure catapulting, which allows the fast isolation of single chromosomes for the generation of chromosome-specific paint probes. To demonstrate the feasibility of this approach, single chromosomes were collected and amplified with degenerate oligonucleotide-primed PCR, hapten-labeled and hybridized onto normal metaphase spreads. Fluorescence in situ hybridization signals revealed specific painting of the respective chromosomes.

Animals↗
PubMed1983

Technical note: a new method for preparing renal tissue for microdissection.

The rat kidney has been prepared for microdissection by in vivo perfusion with hyaluronidase and collagenase and subsequent fixation with glutaraldehyde. The procedure ensures preservation of cytoplasmic detail, and the principle of fixation after maceration should provide a foundation for developing a variety of staining and histochemical studies.

Animals↗
PubMedDate not supplied

Biochemical analysis of microdissected sections from the normal and cataractous human lens.

To better compare possible changes in soluble protein content during human cataractogenesis, microdissected sections from opaque and highly pigmented versus transparent parts of the lens were analyzed by high pressure liquid chromatography, isoelectric focusing, sodium dodecylsulfate polyacrylamide gel electrophoresis, and tryptic mapping of radioiodinated peptides. Using this methodology, resolution of proteins and their tryptic peptides can be obtained from individual lens sections containing as little as 5-10 micrograms of protein. Furthermore, the origin of each protein analyzed by these methods can be verified by comparison with tryptic peptide maps of previously purified human crystallins. Included are examples of how this technique can be used to characterize differences in protein composition from opaque versus transparent sections from different human lenses, as well as brunescent vs lightly-pigmented sections from the same human lens.

Autoradiography↗
PubMed1987

Preparation of retinal vessels for scanning electron microscopic examination using microdissection by ultrasonication.

A method of microdissection by ultrasonication was used to prepare blood vessels of the retina for scanning electron microscopic examination. Eye cups were treated with 2% OsO4 that contained 2% CHAPS or 1% saponin (16 hrs.), dehydrated to 100% acetone, sonicated at 80 kHz (40 min.), and further processed by conventional means. This treatment resulted in the separation of the outer and inner retina at the level of blood vessels in the outer plexiform layer. The overall pattern of the blood vessels was maintained. Venules were distinguished from capillaries. This technique may provide information that is not possible to obtain with corrosion casts or trypsin digests.

Animals↗
PubMed1989

Levels of immunoreactive angiotensin II in microdissected nuclei from adult WKY and SH rat brain.

Levels of immunoreactive angiotensin II (ANG II) were measured by radioimmunoassay in microdissected nuclei from the brain of the spontaneously hypertensive rat (SHR) and its normotensive control, the Wistar Kyoto rat (WKY). The nuclei assayed included the paraventricular nucleus of the hypothalamus (PVH), locus coeruleus (LC), nucleus of the solitary tract (NTS), dorsal motor nucleus of the vagus nerve (DMN of X) and the A1 region of the medulla. Sections of cerebral and cerebellar cortex were used as controls. Levels of immunoreactive ANG II ranged from 0.24 nmoles/g protein to 0.93 nmoles/g protein, with SHR brain containing significantly higher levels than WKY brain in the PVH and NTS. Although LC and DMN of X in the SHR brain contained higher levels of immunoreactive ANG II than WKY brain, the difference was not significant. No significant difference was found in the A1 region between the two species, and no detectable ANG II immunoreactivity was found in cerebral or cerebellar cortex. Since each of the nuclei studied has been implicated in the neural control of cardiovascular function, the increased levels of immunoreactive ANG II in the SH rat brain nuclei indicates a possible role for ANG II in the pathophysiology of hypertension in the SHR model.

Angiotensin II↗
PubMed1986

Intestinal plexuses in Crohn's disease and ulcerative colitis in children: pathologic and microdissection studies.

The relative fraction of neural tissue in the plane of the myenteric (Auerbach) plexus (MEP) of ileum and colon of patients with Crohn's disease and ulcerative colitis was analyzed by point-count morphometric analysis of microdissected flat-mount ("circuit diagram") preparations of the plexus. Increase in the relative fraction of neural tissue (myenteric plexus hyperplasia) was seen in the MEP of some patients in both ileum and colon in both diseases. The frequency of ulceration or inflammation extending into the bowel wall deep enough to injure the various levels of the intestinal plexus system (mucosal plexus of Cajal; upper submucosal plexus of Meissner; deeper submucosal plexus of Henle; intermuscular plexus of Auerbach; outer muscular/serosal plexus of Stöhr) was analyzed for small intestine, appendix, and colon for patients with both diseases. As expected, Crohn's disease tends to produce deeper intestinal wall damage in ileum versus colon, and ulcerative colitis the reverse. The effect of such injury on local and more distal motor control of the intestine and on integrative functions of the intestinal plexus system appears not to have been considered previously. Since injury deep enough to damage any given plexus necessarily damages all overlying plexus levels, analysis of such effects appears a fruitful field for study.

Adolescent↗
PubMed1986

Microdissection study of the incidence of branched eccrine sweat glands and the number of eccrine glands per unit area of infants' and childrens' skin.

That branched eccrine sweat glands occur has rarely been mentioned in the literature, and their incidence has never been determined. All eccrine glands in 0.5-cm2 pieces of anterior trunk skin of 59 children were microdissected, and the total number of glands and the number of branched eccrine glands determined. One-hundred fourteen of 17,539 (0.65%) were branched, 90% in the middermis and 10% at the epidermis. The apparently normal anatomic property that almost 1% of eccrine sweat glands are branched has not hitherto been appreciated. One doubly branched gland was found. Patients with leukemia (11 in the study) possibly had more branched glands than nonleukemic patients of the same body size. The best statistical relation of the number of sweat glands per unit area of skin (GUA) to surface area (SA) or age in children was the natural logarithm of GUA versus the reciprocal of surface area: LnGUA = (0.2205 X 1/SA) + 5.42. This result is consistent with the classical proposition that there is no important degree of formation of new eccrine glands, nor of loss, after birth, the density of glands per unit area of skin reducing as SA rises with growth during childhood.

Child↗
PubMed1986

Microdissection demonstration of the lesion of the endocrine kidney in children.

Microdissection of nephrons of kidneys of children showing advanced ischemic tubular atrophy, and removed for control of hypertension, demonstrates marked proximal convoluted tubular atrophy, with formation of multiple small proximal tubular diverticula. These diverticula presumably contribute to the microscopic appearance of large numbers of small tubules lined by low epithelial cells with pale or clear cytoplasm, adjacent to glomeruli in the cortices of kidneys showing ischemic tubular atrophy (endocrine kidney). Segmentation of such atrophic tubules leads to formation of blind segments (microcysts), as demonstrated in this study and by Oliver. The distinctive microscopic appearance of the endocrine kidney, a not infrequent finding in kidneys of children with chronic renal insufficiency who require nephrectomy for control of hypertension, has not hitherto been emphasized in the literature on pediatric renal disease.

Adolescent↗