PURPOSE: To identify genes that showed altered expression between human polymorphonuclear (PMN) cell cultured on plastic and on amniotic membrane by the technique of differential hybridization of two Altas(TM) Human cDNA expression array. METHODS: 32P-labeled complimentary DNA probes derived from RNA of either human polymorphonuclear leukocyte cultured on plastic and cultured on amniotic membrane were hybridized to two identical human cDNA expression array membranes containing 588 known genes. RESULTS: Of the total 588 genes, 130 genes were up- or down-regulated. 50 up-regulated and 80 down-regulated genes were identified in polymorphonuclear leukocyte cultured on amniotic membrane compared with control. After different signal intensity was normalized more than 4000 by Atlas Image(TM) 1.0 Software, 19 genes were up-regulated and 36 genes down-regulated. CONCLUSIONS: Genes associated with the process of apoptosis, DNA synthesis and repair were down-regulated in PMN cultured on AM and genes associated with DNA binding protein, transcription factor were altered. Cell-cell communication factors including TGF-beta, PDGF-A, RANTES, MRP-14, oncostatin M, MIP-2 alpha were significantly down-regulated and cell surface antigen CD11a (LFA-1) was down-regulated, suggesting that AM can suppress the inflammatory reaction mediated by adhesion molecule, inflammatory, proinflammatory cytokines and chemokines.
Amnion* ; Antigens, Surface ; Apoptosis ; Chemokine CCL5 ; Chemokines ; Cytokines ; DNA ; DNA Probes ; DNA, Complementary ; DNA-Binding Proteins ; Humans* ; Inflammation ; Membranes ; Neutrophils* ; Oligonucleotide Array Sequence Analysis ; Oncostatin M ; Plastics ; RNA ; Transcription Factors ; Transforming Growth Factor beta

In order to identify the differentially expressing gene of bone marrow mesenchymal stem cells (MSCs) stimulated by electromagnetic field (EMF) with osteogenesis microarray analysis, the bone marrow MSCs of SD rats were isolated and cultured in vitro. The third-passage cells were stimulated by EMFs and total RNA was extracted, purified and then used for the synthesis of cDNA and cRNA. The cRNA of stimulated group and the control group was hybridized with the rat oligo osteogenesis microarray respectively. The hybridization signals were acquired by using X-ray film after chemiluminescent detection and the data obtained were analyzed by employing the web-based completely integrated GEArray Expression Analysis Suite. RT-PCR was used to identify the target genes: Bmp1, Bmp7, Egf and Egfr. The results showed that 19 differentially expressing genes were found between the stimulated group and the control group. There were 6 up-regulated genes and 13 down-regulated genes in the stimulated group. Semi-quantitative RT-PCR confirmed that the expressions of Bmp1, Bmp7 mRNA of the stimulated group were up-regulated (P<0.05) and those of Egf, Egfr were down-regulated (P<0.05). It was suggested that the gene expression profiles of osteogenesis of the bone marrow MSCs were changed after EMF treatment. It is concluded that the genes are involved in skeletal development, bone mineral metabolism, cell growth and differentiation, cell adhesion etc.
Bone Marrow Cells/*cytology ; Cell Differentiation ; Down-Regulation ; Electromagnetic Fields ; Gene Expression Profiling ; Gene Expression Regulation ; Mesenchymal Stem Cells/*cytology ; Nucleic Acid Hybridization ; Oligonucleotide Probes/chemistry ; Osteogenesis/*genetics ; RNA, Complementary/metabolism ; Reverse Transcriptase Polymerase Chain Reaction

BACKGROUND: The conventional method for decalcification of bone specimens uses hydrochloric acid (HCl) and is notorious for damaging cellular RNA, DNA, and proteins, thus complicating molecular and immunohistochemical analyses. A method that can effectively decalcify while preserving genetic material is necessary. METHODS: Pairs of bilateral bone marrow biopsies sampled from 53 patients were decalcified according to protocols of two comparison groups: EDTA versus HCl and RDO GOLD (RDO) versus HCl. Pairs of right and left bone marrow biopsy samples harvested from 28 cases were allocated into the EDTA versus HCl comparison group, and 25 cases to the RDO versus HCl comparison group. The decalcification protocols were compared with regards to histomorphology, immunohistochemistry, and molecular analysis. For molecular analysis, we randomly selected 5 cases from the EDTA versus HCl and RDO versus HCl groups. RESULTS: The decalcification time for appropriate histomorphologic analysis was the longest in the EDTA method and the shortest in the RDO method. EDTA was superior to RDO or HCl in DNA yield and integrity, assessed via DNA extraction, polymerase chain reaction, and silver in situ hybridization using DNA probes. The EDTA method maintained intact nuclear protein staining on immunohistochemistry, while the HCl method produced poor quality images. Staining after the RDO method had equivocal results. RNA in situ hybridization using kappa and lambda RNA probes measured RNA integrity; the EDTA and RDO method had the best quality, followed by HCl. CONCLUSIONS: The EDTA protocol would be the best in preserving genetic material. RDO may be an acceptable alternative when rapid decalcification is necessary.
Biopsy* ; Bone Marrow* ; Decalcification Technique ; DNA ; DNA Probes ; Edetic Acid ; Humans ; Hydrochloric Acid ; Immunohistochemistry ; In Situ Hybridization ; Nuclear Proteins ; Polymerase Chain Reaction ; RNA ; RNA Probes ; Silver

BACKGROUND: A practical RNA in situ hybridization method using digoxigenin labeled RNA probes is described in order to evaluate the technical difficulties and problems in RNA in situ hybridization. METHODS: The paraffin sections, routinely processed in the Pathology Laboratory, were tested for the possibility of RNA in situ hybridization instead of the RNase free paraffin sections, fixed in 4% paraformaldehyde and prepared using RNase protection procedures. RESULTS: Most of the paraffin sections, fixed in 10% neutral formalin solution in fresh condition, showed relatively good reaction of RNA in situ hybridization, although the necrotic tissue and autopsy specimens showed poor reaction of RNA in situ hybridization. A refixation procedure using a 4% paraformaldehyde solution was evaluated for optimal expression of mRNA in the paraffin sections. CONCLUSION: The treatment of 4% paraformaldehyde before the treatment of proteinase K showed better in situ hybridization than did the treatment of 4% paraformaldehyde after the treatment of proteinase K. Also a new Polymerase Chain Reaction (PCR)-based method of RNA probe production showed consistently good results.
Autopsy ; Digoxigenin* ; Endopeptidase K ; Formaldehyde ; In Situ Hybridization* ; Paraffin ; Pathology ; Polymerase Chain Reaction ; Ribonucleases ; RNA Probes ; RNA* ; RNA, Messenger

OBJECTIVETo study the expression pattern of cd99l2 gene during zebrafish development, the RNA probes for whole-mount in situ hybridization were prepared in this study.

METHODSThe cd99l2 fragment obtained by RT-PCR was cloned into pGM-T Easy, then the plasmids were linearized with the restriction enzymes SacII or SalI. Using Sp6 or T(7) RNA polymerase, the digoxingenin-labeled antisense and sense probes were synthesized and confirmed by whole-mount in situ hybridization.

RESULTSThe plasmid cd99l2/pGM-T was constructed. cd99l2 gene expression pattern during embryogenesis of zebrafish was examined using the antisense probe, and intense expression was detected in the central nervous system during zebrafish development.

CONCLUSIONThe antisense probe can be used for study of the spatial and temporal distribution of cd99l2 during zebrafish development using the sense probe as control.

Animals ; Central Nervous System ; embryology ; Cloning, Molecular ; Digoxigenin ; chemistry ; Gene Expression Regulation, Developmental ; Oligonucleotide Probes ; RNA Probes ; Uridine Triphosphate ; chemistry ; Zebrafish ; embryology ; genetics ; Zebrafish Proteins ; genetics

BACKGROUND: The ischemia responsive protein 94 kDa(irp94) gene belongs to the heat shock protein 110 family and was isolated in 1999 from rat brain by transiently induced forebrain ischemia. The PC12 cell is the pheochromocytoma cell line of rat, which is differentiated to a sympathetic neuron-like cell by the stimulation of a nerve growth factor. This study is to determine whether irp94 is expressed when an ischemia-like condition is induced by ATP depletion in cultured PC12 cells in vitro. METHODS: PC12 cells were maintained as monolayer cultures in RPMI-1640 medium(Sigma) supplemented with 10% horse serum, 5% fetal bovine serum, 5 mg/ml transferrin, and 1 mg/ml insulin in a humidified 5% CO2 incubator at 37degrees C. The ATP depleting agent antimycin A was added at concentrations of 1, 2.5, and 5 microM to simulate ischemia, and 10 microgram/ml of tunicamycin, which is expected to express heat shock protein maximally, was used as a positive control. The cells were harvested after a 60-minute incubation, and the total RNA was extracted. The reverse transcription polymerase chain reaction(RT-PCR) was performed to use 501 bp irp94 cDNA as a molecular probe, and the expression of irp94 mRNA was analyzed by northern blotting. RESULTS: The irp94 mRNA expression was enhanced, compared to the negative control group, as the concentration of antimycin A was increased. CONCLUSION: This study suggests that irp94 mRNA expression is enhanced as the severity of ischemia is increased. Thus, it is possible to investigate the mechanism of ischemic neuronal injury indirectly by using this in-vitro model of neuronal ischemia.
Adenosine Triphosphate ; Animals ; Antimycin A ; Blotting, Northern ; Brain ; Cell Culture Techniques* ; DNA, Complementary ; Gene Expression* ; Heat-Shock Proteins ; Horses ; HSP110 Heat-Shock Proteins ; Humans ; Incubators ; Insulin ; Ischemia* ; Molecular Probes ; Nerve Growth Factor ; Neurons* ; PC12 Cells ; Prosencephalon ; Rats ; Reverse Transcription ; RNA ; RNA, Messenger ; Transferrin ; Tunicamycin

BACKGROUND: Proliferative lesions of the stomach were investigated by in situ hybridization using RNA probes for telomerase components and compared with the results by TRAP (telomeric repeat amplification protocol) assay. METHODS: RNA probes for hTR (human telomerase RNA component) and hTERT (mRNA coding for a catalytic subunit of human telomerase) were made by cloning and in vitro transcription. The probes were applied for in situ hybridization in 23 cases of adenocarcinoma of the intestinal type and adjacent dysplasia, and in the normal and metaplastic mucosa of the stomach. RESULTS: Telomerase activity by TRAP was positive in all cases of adenocarcinoma, most cases of dysplasia, and many cases of normal mucosa. hTR in situ hybridization showed positive staining in the adenocarcinoma cells, dysplastic cells, a few cells in the proliferation zone of the normal mucosa, and a few infiltrated lymphocytes. hTERT showed positive staining in the same cells. CONCLUSIONS: Telomerase is expressed in most cases of dysplastic lesions and is thought to be acquired in the early steps of carcinogenesis. The expression is noted in a few cells of the normal proliferative zones and the infiltrated lymphocytes, emphasizing the importance of in situ detection of telomerase at the cell level.
Adenocarcinoma ; Carcinogenesis ; Catalytic Domain ; Clinical Coding ; Clone Cells ; Cloning, Organism ; Humans* ; In Situ Hybridization ; Lymphocytes ; Mucous Membrane ; Polymerase Chain Reaction ; RNA Probes ; RNA* ; RNA, Messenger* ; Stomach* ; Telomerase*

PURPOSE: The control of tuberculosis is seriously threatened worldwide by the recently emerging multidrug-resistant Mycobacterium tuberculosis. As a result, early detection of drug resistant M.tuberculosis strain has become very important but conventional laboratory methods are time consuming and delayed results often affect patients adversely in controlling tuberculosis. The authors studied the usefulness of the line probe assay to determine the mutaion in rpoB gene of rifampin resistant M.tuberculosis and to find out if this method can substitute conventional methods in the detection of resistant strain. METHODS: This study employed 40 clinical samples of M.tuberculosis which had been determined by culture and drug sensitivity test. After amplification of rpoB-the gene for the B subunit of the RNA polymerase-by PCR, the amplified products were hybridized with specific oligonucleotide probes immobilized on nitrocellulose strip and direct DNA sequencing was also performed. The results were compared with those of the classical susceptibility test. RESULTS: Among the 40 samples, 10 were identified as drug resistant strain by classical drug susceptibility test. Three of the ten resistant samples were rifampin resistant strains, which were identified by either method. All mutations were clustered within the region of 69bp of rpoB and all were single nucleotide mutations. Two isolates had a TCG->TTG(serine->leucine) mutation in codon 522. One isolate had a CAC->CTC(histidine->leucine) mutation in codon 526. CONCLUSION: In contrast to culture and sensitivity tests, line probe assay is an easy and speedy method for detecting rifampin resistant M.tuberculosis in clinical samples as well as a helpful tool for choosing antituberculosis drug in children.
Child ; Codon ; Collodion ; DNA-Directed RNA Polymerases* ; Early Diagnosis* ; Humans ; Mycobacterium tuberculosis* ; Mycobacterium* ; Oligonucleotide Probes ; Polymerase Chain Reaction ; Rifampin ; RNA Polymerase II* ; RNA* ; Sequence Analysis, DNA ; Tuberculosis

BACKGROUND: A presumptive histopathologic diagnosis of tuberculosis is commonly based on the finding of acid- fast bacilli upon microscopic examination of a diagnostic specimens. Although this traditional histochemical staining methodis satisfactory, it is time-consuming and not species-specific. For more specific assessment, in situ hybridization assay with oligonucleotide probes is introduced. METHODS: The human surgical specimens were obtained from tuberculosis patients(,)and experimental specimens were made by injecting fresh rat liver with cultured M. tuberculosis organisms into fresh rat liver. Oligonucleotide probes complementary to ribosomal RNA portion were synthesized and labeled with multiple biotin molecules. For a rapid detection, all procedures were carried out using manual capillary action technology on the Microprobe staining system. RESULTS: The in situ hybridization assay produced a positive reaction in experimental specimens (80-90% sensitivity) after pepsin- HCl pre-treatment for a good permeabilization of probes, but reliable result was not obtained from human surgical specimens. CONCLUSION: It is, therefore, suggested that biotin- labeled oligonucleotide probes have considerable potential for identification and in situ detection of M. tuberculosis but, there are some barriers to overcome for the diagnostic use of this method.
Animals ; Biotin ; Capillary Action ; Diagnosis ; Humans ; In Situ Hybridization* ; Liver ; Mycobacterium tuberculosis ; Oligonucleotide Probes ; Rats ; RNA, Ribosomal ; Tuberculosis*

Recently protective and supportive functions of neurotrophic factors on dopaminergic neurons have been reported. In this study, in situ hybridization histochemistry with "S-labeled oligonucleotide probes for brain-derived factor (BDNF) and neurotrophin-3 (NT-3) mRNAs was performed to determine the effect of unilateral deafferentation of midbrain dopaminergic cells with 6-hydroxydopam'me (6-OHDA) on the expression of niRNAs of the above neurotrophic factors in the hippocampal areas. The deafferentation of midbrain dopaminergic cells induced changes of expression of BDNF mRNAs and NT-3 mRNAs. Although the reduction of NT-3 MRNA is limited to dentate gyrus of the lesion side, the induction of BDNF MRNA was observed in the lesion side firstly and then showed in the contralateral side conseqently. These results support the suggestion that these neurotrophic factors may protect or support dopaminergic neurons. In addition, these data propose the possibility that neurotrophic factors may be related with degenerative diseases such as Parkinson's disease.
Brain-Derived Neurotrophic Factor ; Dentate Gyrus ; Dopaminergic Neurons ; In Situ Hybridization ; Mesencephalon ; Nerve Growth Factors* ; Oligonucleotide Probes ; Parkinson Disease ; RNA, Messenger*