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PubMed2003

Flow cytometric sorting of paraffin-embedded tumor tissues considerably improves molecular genetic analysis.

The characterization of genetic aberrations in paraffin-embedded tumor material is impaired by contaminating normal cells. In the present study on the genetic causes of loss of HLA expression in diffuse large B-cell lymphoma (DLBCL), we compared the efficacy of microdissection with flow cytometric sorting of tumor cells. Single-cell suspensions from paraffin-embedded material of 5 DLBCL cases were stained for CD79a and DNA content (propidium iodide). Fluorescent in situ hybridization (FISH) using HLA class II and chromosome 6 centromeric probes and loss of heterozygosity (LOH) analysis with 5 HLA-specific microsatellite markers were performed on microdissected and flow cytometry-sorted fractions. FISH confirmed considerable enrichment of the samples after flow cytometric sorting and disclosed tumor heterogeneity in 4 cases. Moreover, lymphomas with a so-called zebra LOH pattern in the microdissected material showed unambiguous LOH after flow cytometric sorting, revealing in 1 case a biologically relevant hemizygous deletion in the HLA region.

Chromosome Aberrations↗
PubMed1994

Prolactin- and testosterone-induced inhibition of LH secretion after orchidectomy: role of preoptic and tuberoinfundibular gamma-aminobutyric acidergic neurones.

The inhibitory amino acid neurotransmitter gamma-aminobutyric acid (GABA) may play an important role in the regulation of LH-releasing hormone secretion. The present study examined the effect of prolactin on GABAergic neuronal activity in microdissected brain regions of the orchidectomized rat, to determine whether inhibition of LH secretion after castration by acute hyperprolactinaemia was associated with prolactin-induced changes in GABAergic neuronal activity. The effects of prolactin were contrasted with the effects of testosterone on hypothalamic GABAergic neurones after orchidectomy. GABA concentrations were measured by high pressure liquid chromatography in eight microdissected brain regions in untreated rats and 60 min after inhibition of the GABA catabolic enzyme GABA transaminase by injection of amino-oxyacetic acid (AOAA). The rate of GABA accumulation in microdissected brain regions following injection of AOAA was taken as an index of GABAergic neuronal activity. Rats were divided into seven experimental groups: intact controls, 2 days after castration, 2 days after castration with prolactin treatment (2.5 mg ovine prolactin injected s.c. every 12 h, starting at the time of castration), 2 days after castration with testosterone replacement (30 mm silicone elastomer implant containing crystalline testosterone), 6 days after castration, 6 days after castration with prolactin treatment, and 6 days after castration with testosterone replacement. Both 2 and 6 days after castration, plasma LH was markedly elevated above levels in intact rats, and AOAA-induced GABA accumulation was significantly decreased in the diagonal band of Broca at the level of the organum vasculosum of the lamina terminalis, in the medial preoptic nucleus and in the median eminence. Hyperprolactinaemia significantly reduced LH levels 2 days but not 6 days after castration. GABAergic neuronal activity, however, was not significantly affected by prolactin at either time. Testosterone replacement blocked the postcastration elevation in plasma LH and prevented the castration-induced suppression of GABAergic neuronal activity both 2 and 6 days after castration. There were no castration- or hormone-induced changes in GABAergic neurones observed in the medial or lateral septum, caudate nucleus, cingulate cortex or arcuate nucleus. These results demonstrate that the activity of GABAergic neurones terminating in the rostral hypothalamus and the median eminence is positively regulated by testosterone, and that these steroid-sensitive GABAergic neurones may be important in the negative-feedback control of LH secretion. Inhibition of LH secretion by hyperprolactinaemia, however, may not be mediated by changes in GABAergic neuronal activity.

Animals↗
PubMed2000

Processing renal biopsies for diagnostic mRNA quantification: improvement of RNA extraction and storage conditions.

The goal of this study was to improve a procedure for the extraction and storage of RNA from minute quantities of human renal tissue in clinical practice, using kidney biopsies and cadaveric donor kidneys unsuitable for transplantation. Collagen alpha1(IV) mRNA was analyzed as a measure for RNA integrity. The results show that at least 3 h may pass between microdissecting the renal tissue and the onset of cDNA synthesis without degradation of the glomerular mRNA. To extract the glomerular mRNA, microdissected glomeruli were incubated in a permeabilization solution. Treating glomeruli with collagenase IV before permeabilization had a deteriorating effect on the mRNA yield. The addition of reverse transcription mixture to the permeabilization solution in the presence of the glomeruli resulted in the highest cDNA yields. Storage of glomerular tissue in the presence of Nonidet P-40-based buffer for 1 wk at -70 degrees C did not significantly affect the mRNA, but storage for 2 or 4 wk resulted in deterioration of the mRNA by approximately 40 and 95%, respectively. Furthermore, three methods for total RNA isolation from microdissected interstitial tissue were compared. An approximately 2.5 times higher yield of collagen alpha1(IV) mRNA was obtained with silica gel-based membrane spin technology than with a guanidine isothiocyanate/phenol chloroform or a lithium chloride/phenol chloroform method. Finally, this study shows for the first time reliable detection of collagen alpha1(IV) mRNA in biopsies that had been frozen for at least 10 yr at -70 degrees C. These experiments have helped to improve a procedure for the processing of glomerular and interstitial tissue acquired from human kidney biopsies for mRNA analysis. This method is suitable for implementation in routine clinical practice.

Biopsy↗
PubMed2003

Generation of chromosome paints: approach for increasing specificity and intensity of signals.

Chromosome painting is a widely used technique, and the two principal means of generating probes for such experiments involve DNA isolation by chromosome flow sorting and by chromosome microdissection. Frequently, chromosome paints are bright and specific; however, on occasion, signals can be weak and nonspecific, particularly for microdissected probes. Reasons for this have been attributed to co-amplification of non-target DNA and the formation of primer concatamers during degenerate oligonucleotide primed (DOP)-PCR. Here we describe a technique of circumventing this problem by sequence enrichment. It involves co-hybridization of DOP-PCR biotinylated microdissected material and linkered genomic DNA. Biotinylated DNA fragments captured on streptavidin-coated paramagnetic beads are eluted and amplified by PCR using a single primer complementary to the linker arm.

Animals↗
PubMed1987

Micromethods for the study of GABA biochemistry and function at single GABA acceptive membranes.

Three different micromethods for studying GABA biochemistry and function at single microdissected GABA-acceptive neuronal membranes are discussed. The basis for such studies is the possibility of obtaining by microdissection single Deiters' neurons from the lateral vestibular nucleus of the rat and the rabbit. From these isolated cells the plasma membrane may be prepared and studied. The first micromethod allows the study of the Na+ independent diffusion of GABA through such a plasma membrane which is postsynaptic to GABA-ergic boutons. A modification of such method allows also the study of the effects of GABA-ergic drugs on Cl- permeability. The second method allows the study by microelectrophoresis in capillaries of GABA catabolism by GABA-T associated with microdissected single Deiters' membranes. The third one was developed in order to study the characteristics of Na+ dependent GABA carrier activity present on such membranes.

Animals↗
PubMed1999

In vivo gene expression profile analysis of human breast cancer progression.

The development and use of molecular-based therapy for breast cancer and other human malignancies will require a detailed molecular genetic analysis of patient tissues. The recent development of laser capture microdissection and high density cDNA arrays now provides a unique opportunity to generate gene expression profiles of cells from various stages of tumor progression as it occurs in the actual neoplastic tissue milieu. We report the combined use of laser capture microdissection and high-throughput cDNA microarrays to monitor in vivo gene expression levels in purified normal, invasive, and metastatic breast cell populations from a single patient. These in vivo gene expression profiles were verified by real-time quantitative PCR and immunohistochemistry. The combined use of laser capture microdissection and cDNA microarray analysis provides a powerful new approach to elucidate the in vivo molecular events surrounding the development and progression of breast cancer and is generally applicable to the study of malignancy.

Breast Neoplasms↗
PubMed2000

Concurrent and independent genetic alterations in the stromal and epithelial cells of mammary carcinoma: implications for tumorigenesis.

The high frequency of loss of heterozygosity (LOH) in epithelial cells of mammary ductal carcinoma in situ (DCIS) and IDC is a well known phenomenon, whereas the genetic abnormalities in the mammary stroma and its influence on the epithelial component have not been sufficiently studied. Using the PCR, we examined DNA extracts from microdissected stromal and epithelial tissues of 11 breast samples containing DCIS, including five cases associated with IDC. In each case, the mesenchymal tissue consisting of normal-appearing stroma at a distance from DCIS and IDC or stroma close to either DCIS or IDC was manually microdissected. Epithelial cells from morphologically clear-cut normal ducts and lobules, DCIS, and IDC were also microdissected. Twelve polymorphic DNA markers were tested to identify possible genetic alterations in the mesenchymal and epithelial cells on chromosomes 2p, 3p, 11q, 16q, and 17q. Samples from bilateral reduction mammoplasty from 10 women without any clinical, radiological, or pathological abnormalities were also selected as a control (reduction mammoplasty group). Whereas most cases (8/11, 73%) displayed at least one identical LOH in both epithelial and mesenchymal components, LOH at several loci was noted exclusively in stromal cells. The most frequent genetic alterations in the mesenchymal cells were at chromosomes 17q24, 16q23.1-24.2, 3p14.2, and 11q21-23.2, in 87.5, 62, 60, and 45% of informative cases, respectively. The LOH frequency in the stroma close to cancer ranged from 10 to 66.5% for DCIS and from 20 to 75% of informative cases for IDC. Furthermore, 10 of the 12 polymorphic markers revealed LOH in the stroma at a distance, ranging from 11 to 57% of informative cases. None of the control cases (women without any breast disease) revealed LOH either in the epithelial or in the stromal components. Our findings strongly support the concept of stromal-epithelial interaction in the development and progression of mammary neoplasia. Furthermore, this study suggests that genetic alterations in the stromal cells may precede genotypic changes in the epithelial cells. At least in some cases, the mammary stroma in DCIS or IDC apparently represents a neoplastic interactive component rather than a reactive response to the carcinoma. The frequent allelic loss (LOH) in the mammary stroma, identified in our study, may explain some of the fibroblastic abnormalities previously observed in patients with breast carcinoma or a variety of cancer-associated hereditary diseases. We conclude that the mammary stroma may play a key role in inducing neoplastic transformation of epithelial cells, recapitulating its role in normal mammary duct development.

Breast Neoplasms↗
PubMed2000

[An approach to early genetic alterations in precancerous cells].

To investigate the potential role of the PTEN tumor-suppressor gene in the carcinogenesis of ovarian endometrioid carcinoma and its related subtype, clear cell carcinoma, we examined 20 ovarian endometrioid carcinomas, 24 clear cell carcinomas and 34 solitary endometrial cysts of the ovary for LOH at 10q23.3 and point mutations of the PTEN gene, using a laser-assisted microdissection method. LOH was found in 8 of 19 ovarian endometrioid carcinomas (42.1%), 6 of 22 clear cell carcinomas (27.3%) and 13 of 23 solitary endometrial cysts (56.5%). Somatic mutations in the PTEN gene were identified in 4 of 20 ovarian endometrioid carcinomas (20.0%), 2 of 24 clear cell carcinomas (8.3%) and 7 of 34 solitary endometrial cysts (20.6%). In 5 endometrioid carcinomas with endometriosis, 3 displayed LOH events common to both the carcinoma and the endometriosis. In 7 clear cell carcinomas with endometriosis, 3 displayed LOH events common to both the carcinoma and the endometriosis. In no cases there were LOH events in the endometriosis only. These results indicate that inactivation of the PTEN gene is an early event in the development of both endometrioid and clear cell carcinoma of the ovary. A laser-assisted microdissection method enables us to collect target cells without contamination by non-tumor cells. We expect that this technique will be very useful for investigating genetic alterations in cancerous or precancerous lesions. Early genetic alterations in various precancerous cells detected by light microscopy can be readily identified by the tissue-microdissection method.

Adenocarcinoma, Clear Cell↗
PubMed2001

Isolation of novel human fetal brain cDNAs mapped to human chromosome bands, 1q25 and 8q24.1.

We isolated chromosome band-specific human fetal brain cDNAs by the microdissection mediated cDNA capture method, and localized these cDNA using in situ hybridization histochemistry with developing rat brain sections. Uni-Amp cDNAs were prepared from an 18-week old human fetal brain, and hybridized to human metaphase chromosomes. Eight Uni-Amp cDNAs, hybridized to chromosome band 1q25 or 8q24.1, were recovered by microdissection and PCR amplification with Uni-Amp primers. Among these cDNAs, two novel genes (FB113 of 8q24.1 and FB134 of 1q25) showed a temporospatially interesting expression pattern in the developing rat brains. The expression of FB113 was under dynamic regulation in the developing granule cells of cerebellum and dentate gyrus. FB134 showed a nervous tissue specific expression pattern and an exclusively prominent expression in the developing presubiculum and parasubiculum. By the fluorescence in situ hybridization using human genomic DNAs, FB113 and FB134 were mapped back to the human chromosome bands 8q24.1 and 1q25, respectively. These results indicate that combined application of the microdissection mediated cDNA capture method and in situ hybridization histochemistry can be used for the isolation of chromosomal band-specific genes related to brain development or human genetic diseases.

Animals↗
PubMed2001

Sampling the intramyocellular triglycerides from skeletal muscle.

To determine the extent and microanatomical distribution of extramyocellular adipocytes associated with skeletal muscle, histological, biochemical, nuclear magnetic resonance proton spectroscopic and microcomputed tomography techniques were employed to analyze skeletal muscle samples from lean and obese Sprague-Dawley rats. Significant amounts of extramyocellular adipocytes were found on the exterior surface of rat gastrocnemius, soleus, and tibialis anterior muscles. The triglyceride content of these exterior adipocytes in these muscle groups was 2- to 3-fold greater than that of the respective intramyocellular triglyceride pool (P = 0.01). Thus, the exterior adipocytes associated with skeletal muscle samples are an abundant source of extramyocellular fat potentially contaminating the intramyocellular triglyceride pool if not carefully and completely removed. On the other hand, no adipocytes were found in the interfascicular space (between muscle bundles) or the intrafascicular space (between muscle fibers) in any of the three rat muscles. The feasibility of and procedures for removing extramyocellular fat by microdissection techniques to obtain pure muscle sample were also evaluated. Complete removal of the extramyocellular adipocytes from rat skeletal muscle, using microdissection with a stereo microscope, was found to be practical and effective. It is concluded that pure muscle samples free of contamination by extramyocellular fat can be obtained, but only if microdissection techniques are utilized.

Adipocytes↗
PubMed2000

[Detection of hepatitis C virus RNA in the tissue of hepatocellular carcinoma by multiple detection system].

OBJECTIVE: Detection of hepatic HCV RNA in hepatocellular carcinoma (HCC) is difficult, since its expression is very low. Several techniques have been established. However, false positive and negative rates still exist. In this study, we applied several conventional and recently developed detection systems to determine the exact effect of HCV RNA on the development of RCC. METHODS: Immunohistochemistry for HCV core antigen and in situ hybridization for HCV RNA had been performed in 39 cases of HCC. IS-RT-PCR was applied for detection and localization of HCV RNA. We extracted microdissected liver tissues to detect HCV RNA separately in cancerous and pericancerous tissues so that the microscopic origin of the amplicons could be controlled. The serological tests including ELISA and RT-PCR for HCV also were performed in all cases. RESULTS: The positive rate by ELISA (30.8%) was not always consistent with that by RT-PCR(43.6%)from serum samples. The latter was much more sensitive and accurate to reflect existence of HCV RNA. Immunohistochemistry showed expression of RCV core antigen in 15 of 39 HCC cases. In pericancerous tissues, the signals were mainly localized in the cytoplasm of hepatocytes. However, translocated expression of HCV core protein was observed in the nuclei of tumor cells, the translocated rate was 73.3%. According to IS-RT-PCR, the positive signals were located mainly in the cytoplasm of cancer cells, the positive rate in HCC was 53.8%, expression of HCV RNA, serum HCV RNA level was detected to be low or negative. This suggested that serum HCV RNA was not always a good reflection of hepatic HCV RNA in HCC tissues. The microdissection RT-PCR described here gave an equal positive rate (59.0%) of HCV RNA in cancerous and pericancerous tissues. CONCLUSIONS: The high detection rate of HCV RNA in HCC specimens even from seronegative patients confirms the important role of HCV RNA during malignant transformation. IS-RT-PCR is a good detection and localization method to visualize HCV RNA in HCC tissues. The microdissection RT-PCR method has a distinct advantage and we anticipate this method will provide an important evidence to determine whether HCV play a direct or indirect role in hepatocarcinogenesis.

Adult↗
PubMed2002

Proteomic analysis and identification of new biomarkers and therapeutic targets for invasive ovarian cancer.

Epithelial ovarian cancer kills almost 16 000 women each year in part due to late stage of presentation and lack of reliable biomarkers for disease detection. CA-125, the currently accepted serum marker, alone lacks the sensitivity for early stage diagnosis, as only 50% of early stage cases are detected with this marker. Although more early stage cases may be detected by lysophosphatidic acid, this marker is also elevated in other cancers. One major objective of the NCI-FDA Tissue Proteomics Initiative has been to combine the technique of laser capture microdissection (LCM) of epithelial tumor cells in human tissue specimens with two-dimensional gel electrophoresis (2-D PAGE) to identify proteins that may serve as invasive ovarian cancer-specific biomarkers for early detection and/or new therapeutic targets. We performed 2-D PAGE on lysates from five microdissected ovarian tumors (three invasive ovarian cancers and two noninvasive, low malignant potential (LMP) ovarian tumors). We then compared silver stained 2-D gels created from microdissected lysates with SYPRO-Ruby stained 2-D PAGE profiles of the patient-matched undissected bulk tumor lysates from all five patients. Twenty-three proteins were consistently differentially expressed between both the LMP and three invasive ovarian tumors in the limited study set. Thirteen were uniquely present in all three of the invasive ovarian cancer cases and absent or underexpressed in the two LMP cases. Ten were uniquely present in the LMP cases but absent or underexpressed in all invasive ovarian cancer cases. Credentialing and preliminary target validation of the mass spectrometry identified proteins cut from the Ruby-red stained gels was performed by LCM coupled Western blot and reverse-phase array technology in a study set of six cases (the aforementioned five cases used in the 2-D PAGE profiling component of the study plus one additional LMP case). The analysis revealed that the 52 kDa FK506 binding protein, Rho G-protein dissociation inhibitor (RhoGDI), and glyoxalase I are found to be uniquely overexpressed in invasive human ovarian cancer when compared to the LMP form of this cancer. The direct comparison of LCM generated proteomic profiles of invasive vs. LMP ovarian cancer may more directly generate important markers for early detection and/or therapeutic targets unique to the invasive phenotype.

Biomarkers, Tumor↗
PubMed2003

[A case of intraocular malignant lymphoma diagnosed by immunoglobulin gene rearrangement and translocation, and IL-10/IL-6 ratio in the vitreous fluid].

BACKGROUND: The diagnosis of primary intraocular lymphoma is difficult in many cases even with conventional cytological tests using vitreous samples. Recently new diagnostic tests, such as microdissection and polymerase chain reaction (PCR) and measurement of cytokines using intraocular samples, have been applied to the diagnosis of the disease. We report here a case where we used the new diagnostic tests and the results aided us to make a diagnosis of intraocular lymphoma. CASE: A 68-year-old woman with an initial diagnosis of bilateral idiopathic uveitis with steroid-resistant vitreous opacities underwent a vitreous biopsy. The cytological examinations of the vitreous samples revealed class III. The microdissection and PCR using the vitreous samples detected IgH rearrangement gene in the third framework (FR3A), the complementary determining region 3 (CDR3) of the VH region and Bcl-2-associated translocation. The interleukin (IL)-10 to IL-6 ratio in the vitreous fluid was greater than 100. Because the results of the examinations strongly suggested intraocular lymphoma, the patient was treated with radiation and chemotherapy. One month after the therapy, however, the patient developed multiple metastatic lesions in the brain. The clinical course of the patient together with the new diagnostic results of examinations led to a diagnosis of intraocular lymphoma. CONCLUSION: A combination of tests, such as conventional cytology, microdissection, and PCR, and cytokine assay using intraocular biopsy samples, is useful to make a diagnosis of intraocular lymphoma.

Aged↗
PubMed2006

[BCL-XL expression and mutation in non-Hodgkin's lymphoma].

The study was aimed to investigate the BCL-XL expression and mutation, and its clinical significance in non-Hodgkin's lymphoma. Lymphoma cells were selectively isolated by laser microdissection. BCL-XL expression from lymphoma tissue and microdissected lymphoma cells was measured by using real-time quantitative reverse transcription-polymerase chain reaction. BCL-XL mutation was analyzed by using direct sequencing of PCR products. The results showed that compared to 15 patients with reactive hyperplasia, BCL-XL was overexpressed in follicular lymphoma (n = 30), both in lymphoma tissue (P = 0.0064) and in microdissected lymphoma cells (P < 0.0001). No significant rise of BCL-XL expression was observed in patients with T-cell lymphoma (n = 24) and diffuse large B cell lymphoma (n = 24). In follicular lymphoma, high BCL-XL level was associated with multiple extranodal involvement (P = 0.0004), elevated lactate dehydrogenase level (P = 0.0019), high-risk international prognostic index (P = 0.0013) and a short overall survival time (P = 0.0451). Mutation analysis revealed one synonymous mutation (Codon 109 ACA-->ACC) in one case of follicular lymphoma patient. It is concluded that BCL-XL expression is closely correlated with progress of follicular lymphoma and prognosis of patients with follicular lymphoma. The value of BCL-XL expression as a prognostic marker in follicular lymphoma should be considered.

Base Sequence↗
PubMed1994

Increased gelatinase A (MMP-2) and cathepsin B activity in invasive tumor regions of human colon cancer samples.

Gelatinase A (MMP-2) and cathepsin B are proteinases which have been proposed to participate in human tumor invasion and metastasis. Precise quantitation of the activity of these enzymes in invading tumors has not been previously described. We utilized a novel tissue microdissection technique to determine levels of enzyme activity in specific microscopic areas of invasive human colon cancer. Tissue specimens smaller than one high power field can be extracted from the samples and analyzed. Increased levels of pro-enzyme and active enzyme forms of gelatinase A (MMP-2) and increased cathepsin B activity were localized in regions of tumor invasion as compared with a matched number of normal epithelial cells from the same patient. Levels of progelatinase B (MMP-9) were also increased in the tumors; however, we did not observe activation of this enzyme. To investigate the mechanism of gelatinase A activation, we amplified DNA of specific microdissected tumor cell populations using polymerase chain reaction. We did not detect a mutation in the activation locus of the enzyme in any of the tumors studied, which suggests that activation may be due to up-regulation of a tumor-associated gelatinase A activating species. Microdissection of frozen tissue sections may prove valuable in the study of proteinases in human tumor invasion as well as in the detection of genetic alterations in human cancers.

Base Sequence↗
PubMed1997

Optimizing DOP-PCR for universal amplification of small DNA samples in comparative genomic hybridization.

The standard comparative genomic hybridization (CGH) protocol relies on availability of macroscopic tumor samples, which do not contain too much interfering normal cells. Recently, CGH after universal amplification of genomic DNA with degenerate oligonucleotide primed PCR (DOP-PCR) has been used to detect genetic aberrations in microdissected tumor specimens. However, owing to the technical difficulties, CGH results of only few microdissected samples have so far been published. We have developed an improved protocol for DOP-PCR, which includes direct incorporation of fluorochrome-conjugated nucleotides into the PCR product. Among the four polymerase enzymes tested. ThermoSequenase gave the best yield, with PCR products ranging from 100-4,000 bp. A two-step PCR-procedure was used, consisting of a preamplification with low stringency conditions followed by amplification in more stringent conditions. The method was first validated by hybridizing DOP-PCR-amplified normal DNA against nick-translated reference DNA, which showed uniform amd even hybridization result for all chromosomes. Comparison of DOP-PCR CGH to conventional CGH in MCF-7 breast cancer cell line further indicated that genetic aberrations can be reliable detected after DOP-PCR amplification. The sensitivity of the DOP-PCR-CGH was tested by serial dilution of MCF-7 DNA. Fifty picograms of sample DNA (corresponding roughly to two MCF-7 cells) was sufficient for high quality CGH. Experiments with cells microdissected from intraductal breast cancer demonstrated that carcinoma cells from 1 to 2 ducts were sufficient for a successful DOP-PCR CGH analysis. We conclude that the improved DOP-PCR-CGH protocol provides a powerful tool to study genetic aberrations in different histological subpopulations of malignant as well as precancerous lesions. DOP-PCR also improves the success rate of conventional paraffin-block CGH, because a poor quality or a too low yield of extracted DNA can be compensated by universal DNA amplification by DOP-PCR.

DNA, Neoplasm↗
PubMed1998

[Chromosome assignment of a novel microsatellite polymorphic marker (D14S1435) by fluorescence in situ hybridization].

OBJECTIVE: Chromosome assignment of a novel microsatellite marker (D14S1435) containing CA repeats and showing 11 alleles and 0.85 of PIC value in Chinese population) isolated from microdissected human chromosome 14q24.3 DNA library by fluorescence in situ hybridization(FISH). METHODS: The polymorphic marker was used as probe to screen a human Lambda/DASH genome library, a positive clone was isolated and the recombinant phage DNA was extracted as primary material. The insert was recovered through BamH I digestion to complete and low melting point agarose gel electro- phoresis, after processed with Sau3A I the probe was prepared by linker- catch PCR method and the following fluorescence in situ hybridization(FISH) was conducted to metaphase chromosomes. RESULTS: this novel STR was precisely mapped to human chromosome 14q24.3, and this demonstrated the validity of isolating chromosome band-specific genetic markers from probe pool made by chromosome microdissection. CONCLUSION: Confirmed by chromosome fluorescence in situ hybridization, the novel STR obtained by chromosome microdissection, PCR and subcloning method was really from the micro- dissected region. This not only increases the numbers of genetic markers used for linkage analysis in the region,but also provides a novel valuable genetic marker for the gene diagnosis and gene cloning of the possible disease-causing genes located in the region of 14q24.3.

Chromosome Mapping↗
PubMed1998

Lateral and anterior view to tensor fold and supratubal recess.

HYPOTHESIS: The aim of this study was to find suitable methods for basic anatomic evaluation of the supratubal recess and the anterior surface of the tensor fold. BACKGROUND: The current method of superior microdissection via the middle fossa floor provides a good picture of the anatomy and pathology of the epitympanum, but the supratubal recess can be evaluated only after excision of the tensor fold. Postinflammation changes cannot be examined accurately because destruction of the tensor fold necessarily alters the anatomic details. METHODS: Eight temporal bones were studied via a lateral and 14 via an anterior approach, both complemented by the superior microdissection. Data on 51 earlier superior dissections were reevaluated as to the state of the supratubal recess. Histology was documented from eight biopsy specimens and of four serially sectioned temporal bones, two normal and two infected. RESULTS: The lateral route offered a good view to the tensor tendon and lower portion of the tensor fold, but the anterior malleal ligament obstructed the view to the fold's upper portion. The anterior route offered excellent visibility to the anterior pouch, mesotympanum, tensor fold, and the whole supratubal recess. The tensor fold was mostly fixed superiorly to the bony roof with a narrow or broad soft band of composite tissue, infrequently to the transverse crest directly. Inflammatory changes spread from the tympanic isthmus region to the supratubal space over the fold and, if extensive, formed broad inflammatory and scar tissue bands between the fold and the anterior bony wall. CONCLUSIONS: The supratubal recess and the mesotympanum can best be evaluated via the anterior approach, which should be added to the temporal bone microdissection program. It serves well as the starting route, followed by the conventional superior dissection of the epitympanum. The knowledge gained is indispensable in surgery for chronic otitis media for creation of a large common middle ear air space and functioning aeration pathways.

Culture Techniques↗