An optimized protocol using Steedman’s wax for high-sensitivity RNA in situ hybridization in shoot apical meristems and flower buds of cucumber

In situ mRNA hybridization(ISH)is a powerful tool for examining the spatiotemporal expression of genes in shoot apical meristems and flower buds of cucumber.The most common ISH protocol uses paraffin wax;however,embedding tissue in paraffin wax can take a long time and might result in RNA degradation and decreased signals.Here,we developed an optimized protocol to simplify the process and improve RNA sensitivity.We combined embedding tissue in low melting-point Steedman’s wax with processing tissue sections in solution,as in the whole-mount ISH method in the optimized protocol.Using the optimized protocol,we examined the expression patterns of the CLAVATA3(CLV3)and WUSCHEL(WUS)genes in shoot apical meristems and floral meristems of Cucumis sativus(cucumber)and Arabidopsis thaliana(Arabidopsis).The optimized protocol saved 4–5 days of experimental period compared with the standard ISH protocol using paraffin wax.Moreover,the optimized protocol achieved high signal sensitivity.The optimized protocol was successful for both cucumber and Arabidopsis,which indicates it might have general applicability to most plants.

The Distribution of Repetitive DNAs Along Chromosomes in Plants Revealed by Self-genomic in situ Hybridization

The distribution of repetitive DNAs along chromosomes is one of the crucial elements for understanding the organization and the evolution of plant genomes. Using a modified genomic in situ hybridization （GISH） procedure, fluorescence in situ hybridization （FISH） with genomic DNA to their own chromosomes （called self-genomic in situ hybridization, self-GISH） was carried out in six selected plant species with different genome size and amount of repetitive DNA. Nonuniform distribution of the fluorescent labeled probe DNA was observed on the chromosomes of all the species that were tested. The signal patterns varied among species and were related to the genome size. The chromosomes of the small Arabidopsis genome were labeled almost only in the pericentromeric regions and the nucleolus organizer regions （NORs）. The signals in the relatively small genomes, rice, sorghum, and Brassica oleracea var. capitata L., were dispersed along the chromosome lengths, with a predominant distribution in the pericentromeric or proximal regions and some heterochromatic arms. All chromosomes of the large genomes, maize and barley, were densely labeled with strongly labeled regions and weakly labeled or unlabeled regions being arranged alternatively throughout the lengths. In addition, enhanced signal bands were shown in all pericentromeres and the NORs in B. oleracea var. capitata, and in all pericentromeric regions and certain intercalary sites in barley. The enhanced signal band pattern in barley was found consistent with the N-banding pattern of this species. The GISH with self-genomic DNA was compared with FISH with Cot-1 DNA in rice, and their signal patterns are found to be basically consistent. Our results showed that the self-GISH signals actually reflected the hybridization of genomic repetitive DNAs to the chromosomes, thus the self-GISH technique would be useful for revealing the distribution of the regions where repetitive DNAs concentrate along chromosomes and some chromatin differentiation associated with repetitive DNAs in plants.

Detection of Differentiation Among BB, CC and EE Genomes in the Genus Oryza by Two-probe Genomic in situ Hybridization (GISH)

The genus Oryza consists of two cultivated species (O. sativa L. and O. glaberrima Steud.) and approximately 20 wild relative species widely distributed in the pan-tropics. These species have been classified into four complexes following the Vaughan's taxonomic system([1]). The O. officinalis complex is the largest complex in the genus, which includes ten species, having BE, CC, on, and EE genomes in the diploids as well as BBCC and CCDD genomes in the tetraploids. The relationships among the BE, CC, and EE genomes still remain unclear, although previous studies have indicated certain affinities of these genomes([2-4]). Genomic in situ hybridization (GISH) is a powerful technique to detect the relationships among the related genomes at chromosome and DNA levels. The objective of the present study was to investigate the relationships among the BE, CC and EE genomes in the genus Oryza by the two-probe GISH.

Detection of H.pylori DNA in gastric epithelial cells by in situ hybridization

AIM: To investigate the presence of H.pylori DNA within gastric epithelial cells in patients with H.pylori infection and its possible carcinogenic mechanism. METHODS: Total 112 patients, with pathologically confirmed chronic superficial gastritis, chronic atrophic gastritis, intestinal metaplasia, atypical hyperplasia or gastric cancer were studied. Among them, 28 were H.pylori negative and 84 H.pylori positive. H.pylori DNA in gastric epithelial cells was detected by GenPoint catalyzed signal amplification system for in situ hybridization. RESULTS: In the H.pylori positive group, zero out of 24 chronic superficial gastritis (0.0%), four out of 25 precancerous changes (16.0%) and thirteen out of 35 gastric cancers (37.1%) showed H.pylori DNA in the nucleus of gastric epithelial cells, the positive rates of H.pylori DNA in the nucleus of gastric epithelial cells were progressively increased in chronic superficial gastritis, precancerous changes and gastric cancer groups (chi(2)=12.56, P=0.002); One out of 24 chronic superficial gastritis (4.2%), eleven out of 25 precancerous changes (44.0%) and thirteen out of 35 gastric cancers (37.1%) showed H.pylori DNA in the cytoplasm of gastric epithelial cells (chi(2)=10.86, P=0.004). In the H.pylori negative group, only one patient with gastric cancer was found H.pylori DNA in the nucleus of gastric epithelial cells; Only two patients, one patient with precancerous changes and another with gastric cancer, showed H.pylori DNA in the cytoplasm of gastric epithelial cells. Furthermore, H.pylori DNA must have been in the cytoplasm as long as it existed in the nucleus of gastric epithelial cells. CONCLUSION: H.pylori DNA exists both in the nucleus and the cytoplasm of gastric epithelial cells in patients with H.pylori infections. The pathological progression from chronic superficial gastritis, precancerous changes to gastric cancer is associated with higher positive rates of H.pylori DNA presence in the nucleus of gastric epithelial cells.

MicroRNA-502-3p regulates GABAergic synapse function in hippocampal neurons

Gamma-aminobutyric acid(GABA)ergic neurons,the most abundant inhibitory neurons in the human brain,have been found to be reduced in many neurological disorders,including Alzheimer's disease and Alzheimer's disease-related dementia.Our previous study identified the upregulation of microRNA-502-3p(miR-502-3p)and downregulation of GABA type A receptor subunitα-1 in Alzheimer's disease synapses.This study investigated a new molecular relationship between miR-502-3p and GABAergic synapse function.In vitro studies were perfo rmed using the mouse hippocampal neuronal cell line HT22 and miR-502-3p agomiRs and antagomiRs.In silico analysis identified multiple binding sites of miR-502-3p at GABA type A receptor subunitα-1 mRNA.Luciferase assay confirmed that miR-502-3p targets the GABA type A receptor subunitα-1 gene and suppresses the luciferase activity.Furthermore,quantitative reve rse transcription-polymerase chain reaction,miRNA in situ hybridization,immunoblotting,and immunostaining analysis confirmed that overexpression of miR-502-3p reduced the GABA type A receptor subunitα-1 level,while suppression of miR-502-3p increased the level of GABA type A receptor subunitα-1 protein.Notably,as a result of the overexpression of miR-502-3p,cell viability was found to be reduced,and the population of necrotic cells was found to be increased.The whole cell patch-clamp analysis of human-GABA receptor A-α1/β3/γ2L human embryonic kidney(HEK)recombinant cell line also showed that overexpression of miR-502-3p reduced the GABA current and overall GABA function,suggesting a negative correlation between miR-502-3p levels and GABAergic synapse function.Additionally,the levels of proteins associated with Alzheimer s disease were high with miR-502-3p overexpression and reduced with miR-502-3p suppression.The present study provides insight into the molecular mechanism of regulation of GABAergic synapses by miR-502-3p.We propose that micro-RNA,in particular miR-502-3p,could be a potential therapeutic to rget to modulate GABAergic synapse function in neurological disorders,including Alzheimer's disease and Alzheimer's diseaserelated dementia.

Analysis of the meiosis in the F_1 hybrids of Longiflorum × Asiatic(LA) of lilies(Lilium) using genomic in situ hybridization

Longiflorum and Asiatic lilies of the genus Lilium of the family Liliaceae are two important groups of modem lily cultivars. One of the main trends of lily breeding is to realize introgression between these groups. With cut style pollination and embryo rescue, distant hybrids between the two groups have been obtained. However, the FI hybrids are highly sterile or some of them could produce a small number of 2n gametes, and their BC1 progenies are usually triploids. Dutch lily breeders have selected many cultivars from these BC1 progenies based on their variation. It is presumably suggested that such variation could be caused by intergenomic recombination and abnormal meiosis during gamete formation in F1 hybrids of Longiflorum × Asiatic （LA） hybrids in Lilium. Therefore, the meiotic process of ten F1 LA hybrids was cytologically investigated using genomic in situ hybridization and traditional cytological methods in the present research. The results showed that： at metaphase I, the homoeologous chromosome pairing among different F1 hybrids ranged from 2.0 to 11.4 bivalents formed by homoeologous chromosomes per pollen mother cell （PMC）, and very few multivalents, and even very few bivalents were formed by two chromosomes within one genome rather than homoeologous chromosomes in some PMCs; at anaphase I, all biva- lents were disjoined and most univalents were divided. Both the disjoined bivalents （half-bivalents） and the divided univalents （sister chromatids） moved to the opposite poles, and then formed two groups of chromosomes; because the two resulting half-bivalents retained their axes in the cell undisturbed, many crossover types, including single crossovers, three strand double crossovers, four strand double crossovers, four strand triple crossovers, and four strand multiple crossovers between the non-sister chromatids in the tetrads of bivalents, were clearly inferred by analyzing the breakpoints on the disjoined bivalents. The present investigation not only explained the reason for sterility of the Fl LA hybrids and the variation of their BCx progenies, but also provided a new method to analyze crossover types in other F1 interspecific hybrids as well.

Clinical role and importance of fluorescence in situ hybridization method in diagnosis of H pylori infection and determination of clarithromycin resistance in H pylori eradication therapy

H pylori is etiologically associated with gastritis, gas-tric and duodenal ulcers, gastric adenocarcinoma and mucosa-associated lymphoid tissue (MALT) lymphoma. Eradicating H pylori may convert rapidly the outcome of related diseases with the use of more accurate diagnostic molecular tests. Indeed some of the tests cannot give the evidence of current infection; H pylori can be detected by noninvasive and invasive methods, the latter requiring an endoscopy. Eradication failure is a big problem in H pylori infection. Recently, clarithromycin resistance in H pylori strains is increasing and eradicati-on therapy of this bacterium is becoming more difficult. Molecular methods have frequently been applied besides phenotypic methods for susceptibility testing to detect clarithromycin resistance due to mutations in the 2143 and 2144 positions of 23S rRNA gene. Fluorescence in situ hybridization (FISH) method on paraffin embedded tissue is a rapid, accurate and cost-effective method for the detection of H pylori infection and to determine clarithromycin resistance within three hours according to the gold standards as a non-culture method. This method can also be applied to fresh biopsy samples and the isolated colonies from a culture of H pylori, detecting both the culturable bacillary forms and the coccoid forms of H pylori, besides the paraffin embedded tissue secti-ons. This technique is helpful for determining the bac-terial density and the results of treatment where clarith-romycin has been widely used in populations to increase the efficacy of the treatment and to clarify the treatment failure in vitro.

Chromosome analysis of esophageal squamous cell carcinoma cell line KYSE 410-4 by repetitive multicolor fluorescence in situ hybridization

Chromosome aberrations are distinctive features of human malignant tumors. Analysis of chromosomal changes can illuminate the molecular mechanisms underlying the development and progression of cancer. To establish the technique of multicolor fluorescence in situ hybridization （M-FISH） for identifying chromosome aberrations in esophageal carcinoma cell line KYSE 410-4, four pools of 6-color whole-chromosome painting probes have been designed and hybridized on the same metaphase spread by four rounds of repetitive FISH. Repetitive 6-color M-FISH was successfully established and the cytogenetic abnormalities in KYSE 410-4 cells were characterized. Chromosome gains occurred at 2q, 3, 8, 17p, and X. An isochromosome 3q was visualized in the cell line, which might be one intermediate mechanism leading to 3p losses and/or 3q gains. Furthermore, 16 structural arrangements were detected, including four derivative chromosomes. The rearrangement of the centromeric regions accounted for approximately 44% of all rearrangements. The results added a more complete and accurate information of the genetic alterations to the classical cytogenetic description of KYSE 410-4 and provided a detailed cytogenetic background data for appropriate use of the cell line. The established 6-color M-FISH was useful for analyzing chromosomes in the whole genome of human tumors.

Dual fluorescence in situ hybridization in detection of HER-2 oncogene amplification in primary hepatocellular carcinoma

BACKGROUND: Molecular cytogenetics of oncogene HER-2 amplification in primary hepatocellular carcinoma (HCC) is still unknown. The aim of this study was to in vestigate the frequency of HER-2 oncogene amplification in primary HCC and its relations to clinicopathological pa rameters and prognosis. METHODS: Forty-two surgical samples from patients with primary HCC were detected for their HER-2 oncogene am plification. The number of chromosome 17 and their ratio were tested by dual fluorescence in situ hybridization (FISH) technique, and then the correlations between HER-2 amplification, clinicopathological characteristics and prog nosis were analyzed statistically. RESULTS: HER-2 oncogene amplification was detected in 9 (21.4%) of the 42 primary HCCs, including 4 patient with high copy (HC) (9.5%) and 5 patients with low copy (LC) (11.9%). HER-2 amplification was associated signifi cantly with tumor size and postoperative survival time o HCC patients (P<0.05), and the presence of HER-2 gene amplification was correlated with postoperative relapse (P— 0.257), but not related to sex, age, AFP level, HBV infec tion, histopathological grading and clinical staging of HCC patients (P>0.05). The HER-2 oncogene copy was exa mined in 31 (73.8%) of the 42 primary HCCs, consisting of 9 patients with HER-2 amplification (21.4%) and 22 pa tients with aneuploidy (52.4%). No significant relation were observed between the HER-2 oncogene copy, patien sex, tumor size, histopathological grading, clinical stag ing, postoperative relapse and survival time (P >0.05); bu the HER-2 oncogene copy was correlated significantly to age, AFP level and HBV infection (P <0.05). CONCLUSIONS: There are a lower frequency of HER-2 oncogene amplification and a higher frequency of chromo- some 17 aneuploidy in primary HCC. HER-2 oncogene amplification may be involved in the development and pro- gression of large HCC in some patients, and seems to be a valuably independent prognostic factor predicting the re- currence and poor survival in patients with large HCC.

Cytogenetic comparisons between A and G genomes in Oryza using genomic in situ hybridization

The genomic structures of Oryza sativa （A genome） and O. meyeriana （G genome） were comparatively studied using bicolor genomic in situ hybridization （GISH）. GISH was clearly able to discriminate between the chromosomes of O. sativa and O. meyeriana in the interspecific F1 hybrids without blocking DNA, and co-hybridization was hardly detected. The average mitotic chromosome length of O. meyeriana was found to be 1.69 times that of O. sativa. A comparison of 4,6-diamidino-2-phenylindole staining showed that the chromosomes of O. meyeriana were more extensively labelled, suggesting that the G genome is amplified with more repetitive sequences than the A genome. In interphase nuclei, 9-12 chromocenters were normally detected and nearly all the chromocenters constituted the G genome-specific DNA. More and larger chromocenters formed by chromatin compaction corresponding to the G genome were detected in the hybrid compared with its parents. During pachytene of the F1 hybrid, most chromosomes of A and G did not synapse each other except for 1-2 chromosomes paired at the end of their arms. At meiotic metaphase I, three types of chromosomal associations, i.e.O, sativa-O, sativa （A-A）, O. sativa-O, meyeriana （A-G） and O. meyeriana-O, meyeriana （G-G）, were observed in the F1 hybrid. The A-G chromosome pairing configurations included bivalents and trivalents. The results provided a foundation toward studying genome organization and evolution of O. meyeriana.

Application of fluorescence in situ hybridization in the detection of bladder transitional-cell carcinoma: A multi-center clinical study based on Chinese population

Objective:To evaluate the diagnostic value of fluorescence in situ hybridization(FISH)in bladder cancer.Methods:We enrolled healthy volunteers and patients who were clinically suspected to have bladder cancer and conducted FISH tests and cytology examinations from August 2007 to December 2008.Receiver operating characteristic(ROC)curve analysis was performed and the area under curve(AUC)values were calculated for both the FISH and urine cytology tests.Results:A cohort of 988 healthy volunteers was enrolled to establish a reference range for the normal population.A total of 4807 patients with hematuria were prospectively,randomly enrolled for the simultaneous analysis of urine cytology,FISH testing,and a final diagnosis as determined by the pathologic findings of a biopsy or a surgically-excised specimen.Overall,the sensitivity of FISH in detecting transitional-cell carcinoma was 82.7%,while that of cytology was 33.4%(p<0.001).The sensitivity values of FISH for non-muscle invasive and muscle invasive bladder transitional-cell carcinoma were 81.7%and 89.6%,respectively(p=0.004).The sensitivity values of FISH for low and high grade bladder cancer were 82.6%and 90.1%,respectively(p=0.002).Conclusion:FISH is significantly more sensitive than voided urine cytology for detecting bladder cancer in patients evaluated for gross hematuria at all cancer grades and stages.Higher sensitivity using FISH was obtained in high grade and muscle invasive tumors.

Comparison of Fluorescence in situ Hybridization and Immunohistochemistry for Assessment of HER-2 Status in Breast Cancer Patients

The accurate assessment ofa proto-oncogene, human epidermal growth factor receptor-2 gene （HER-2）, is extremely important for the therapy and prognosis of breast cancer. Currently, immunohistochemistry （IHC） is the method widely used for the detection of HER-2 protein. Fluorescence in situ hybridization （FISH） has been suggested to be a golden standard assay for HER-2 amplification. This study examined the expression and amplification of HER-2 in paraffin-embedded sections of breast cancer tissues, and compared the two methods on the measurement of HER-2 status. HER-2 gene and protein were determined in breast cancer samples from 52 Chinese women by FISH and IHC respectively. The findings indicated that the HER-2 gene amplification was found in 18 cases （34.6%） by FISH and the HER-2 protein over-expression （score 3＋） in 15 cases （28.8%） by IHC. hnmunohistochemically, 28.6% of the cases scored as 2＋ and 93.3% of the cases scored as 3＋ were HER-2-positive by FISH. There was a significant correlation between the HER-2 gene amplification and HER-2 protein over-expression in breast cancer （P〈0.005）. No correlation was noted between the HER-2 gene amplification and any of the clinicopathological parameters examined, including age, menopausal status, menarche age, tumor size, histological tumor type, histological grade, lymph node status, and the expression of ER and PR. It was concluded that the detection of HER-2 gene amplification in breast cancer by FISH is valuable and can compare with HER-2 protein detection by IHC.

A populational survey of 45S rDNA polymorphism in the Jefferson salamander Ambystoma jeffersonianum revealed by fluorescence in situ hybridization(FISH)

The chromosomal localization of 45S ribosomal RNA genes in Ambystoma jeffersonianum was determined by fluorescence in situ hybridization with 18S rDNA fragment as a probe (FISH-rDNA). Our results revealed the presence of rDNA polymorphism among A.jeffersonianum populations in terms of number,location and FISH signal intensity on the chromosomes. Nine rDNA cytotypes were found in ten geographically isolated populations and most of them contained derivative rDNA sites. Our preliminary study provides strong indication of karyotypic diversification of A.jeffersonianum that is demonstrated by intraspecific variation of 45S rDNA cytotypes. rDNA cytotype polymorphism has been described in many other caudate amphibians. We predict that habitat isolation,low dispersal ability and decline of effective population size could facilitate the fixation and accumulation of variable rDNA cytotypes during their chromosome evolution.

Chromosome G-banding in SituHybridization of RFLP Marker umc58 Linked with the Gene hm1 Dictating Helminthosporium carbonum Susceptibility1 in Maize

The technique of simultaneous G banding and in situ hybridization has been developed in plants for the first time.Using this technique.RFLP marker umc58 closely linked with the hm1 gene dictating Helminthosporium carbonum susceptibility1 was localized onto 1L3(chromosome 1,long arm,the third band from the centromere to the end of the arm),5L5 and 9L5.Theresults demonstrated that umc58 was a tripli cated sequence.It was deduced that umc58 probably was in a duplicated region that includes a part of Helminthosporium carbonum susceptibility genes(hm1 and hm2),as the hybridization sites of umc58 in chro mosomes 1 and 9 were those at which the genes localize.The techniques of simultaneous G banding and ISH in plants are discussed.

Analysis of hepcidin expression: In situ hybridization and quantitative polymerase chain reaction from paraffin sections

AIM: To establish methods for quantitative polymerase chain reaction (PCR) for hepcidin using RNAs isolated from paraffin-embedded sections and in situ hybridization of hepatocellular carcinoma (HCC). METHODS: Total RNA from paraffin-embedded sections was isolated from 68 paraffin-embedded samples of HCC. Samples came from 54 male and 14 female patients with a mean age of 66.8 ± 7.8 years. Quantitative PCR was performed. Immunohistochemistry and in situ hybridization for hepcidin were also performed. RESULTS: Quantitative PCR for hepcidin using RNAs isolated from paraffin-embedded sections of HCC was performed successfully. The expression level of hepcidin mRNA in cancer tissues was significantly higher than that in non-cancer tissues. A method of in situ hybridization for hepcidin was established successfully, and this demonstrated that hepcidin mRNA was expressed in non-cancerous tissue but absent in cancerous tissue. CONCLUSION: We have established novel methods for quantitative PCR for hepcidin using RNAs isolated from paraffin-embedded sections and in situ hybridization of HCC.

Niche Differentiation of Phenol-Degrading Microorganisms in UASB Granular Sludge as Revealed by Fluorescence in situ Hybridization

A microbial community structure of granules harvested from an anaerobic sludge blanket reactor treating phenolic wastewater was investigated using fluorescence in situ hybridization(FISH)and clone library construction.Clones of Syntrophorhabdaceae and Cryptanaerobacter were observed to be responsible for phenol degradation.For accurate taxonomic assignment of Cryptanaerobacter clones,phylogenetic analysis using nearly full-length 16S ribosomal RNA(rRNA)gene sequences was necessary.Three oligonucleotide probes were designed to detect the following three taxonomic groups:Syntrophorhabdaceae,Cryptanaerobacter,and Syntrophus.FISH analysis of thin sections of anaerobic granules showed a random distribution of bacteria and archaea.However,a well-defined distribution of Syntrophorhabdaceae,Cryptanaerobacter,and Syntrophus was observed.Cryptanaerobacter and Syntrophus were found on the outer layer of the granules and were closely associated with each other,while Syntrophorhabdaceae was located in the deeper part of the granules.Such specific distribution of the bacteria is most likely due to their metabolic association and affinity for the substrate.Phenol degradation in the granular sludge was observed to be carried out in the following way.First,Cryptanaerobacter converts phenol to benzoate,which is then degraded by Syntrophus into acetate.This syntrophic degradation of phenol occurs near the surface of the granule,where the phenol concen-tration is high.In the deeper part of the granule,where the phenol concentration is lower,Syntrophorhabdaceae degrades phenol into acetate.We observed that Syntrophorhabdaceae is less likely to produce benzoate as an intermediate to feed the neighboring organisms,which contradicts the theo-ries presented by previous studies.

Identification of two Skeletonema costatum-like diatoms by fluorescence in situ hybridization

A harmful algae bloom (HAB) is a dense aggregation of algae in a marine or aquatic environment that can result in significant environmental problems. To forecast the occurrence of HAB, development of a rapid and precise detection method is urgently required. In this study, two Skeletonema costatum-like diatoms (SK-1 and SK-2), were identified morphologically under a light microscope, and detected using fluorescent in situ hybridization (FISH). Strain SK-1 was isolated from a frequently HAB affected area of the East China Sea, and strain SK-2 from an aquatic farm in Qingdao, China. Fluorescent DNA probes were designed that were complementary to the ITS sequence (including 5.8S rDNA) of strain SK-1. After hybridization, strong green fluorescence was observed in cells of strain SK-1 under an epifluorescence microscope; however, no such fluorescence was observed with strain SK-2, which indicates that probes hybridized only the DNA of the target strain, SK-1, in species-specific manner, and that the two strains do not belong to a same species. This finding was confirmed by ITS sequence analysis. The FISH technique used in this study was sensitive, simple, and rapid, and is a promising tool for detecting target HAB species in natural environments.

Development of taxonomic rRNA-targeted probes of two harmful algae: Prorocentrum minimum and Karenia mikimotoi by fluorescence in situ hybridization

Harmful algal blooms recently have been under the spotlight throughout the world, because of their nega- tive impact on the marine environment, aquaculture, fisheries as well as public health. The development of methods for rapid and precise identification and quantification of causative species is essential for the warning and monitoring of blooms, among which the techniques based on taxonomic probes are the most favored. In this study, two harmful algae, i.e., Prorocentrum minimum and Karenia mikimotoi were tak- en into consideration. The partial large subunit rDNA （D1-D2） of both species were firstly PCR-amplified, cloned and sequenced. The obtained sequences were then introduced to carry out alignment analysis for gene specific regions. Three respective candidate probes for each species were designed and used to screen the optimal probe by performing fluorescence in situ hybridization （FISH） tests. The results showed that the probes Pmin0443 and Kmik0602 displayed the best hybridization for P. minimum and K.. mikimotoi, respectively. Both the specific （taxonomic） （Pmin0443 and Kmik0602） and the control probes （UniC0512 and UniR0499） were used for cross-reactivity tests with other microalgae in our laboratory. The probes Pmin0443 and Kmik0602 are specific and could be served as taxonomic probes introduced into the tech- niques targeting rRNA, such as FISH, sandwich hybridization, and DNA-microarray assay of P minimum and K. mikimotoi in the future. Finally, FISH analyses with both probes were performed on the simulated field samples. The probes could hybridize exclusively with the target cells well, and no significant differ- ence （p 〉0.05） was observed in the cell densities of the samples determined by FISH and light microscopy （LM）. All suggest that the probes are specific and could be introduced into FISH for the monitoring of both harmful algae.

DETECTING EXPRESSION OF MRP-1/CD9 mRNA IN LUNG CANCERS USING TISSUE MICROARRAYS AND FLUORESCENCE IN SITU HYBRIDIZATION METHODS

Objective： The aim of this study was to investigate the MRP-1/CD9mRNA expression in lung cancer and normal lung tissues and the relationship between its expression and pathologic grades, clinical stages, metastasis and prognosis. Methods： To observe MRP-1/C9mRNA expression, tissue microarray （TMA） containing 54 lung cancers and 10 normal lung tissues was prepared and Fluorescence in situ hybridization was used. Results： The positive rate of MRP-1/CD9 expression was 48.1% in lung cancer, lower than that of normal lung tissues. The statistical difference was significant （P〈0.05）. Its protein expression had no relationship with the patients＇ ages, sex and the macroscopic type of tumor, but had relationships with the histological type, clinical stage, differentiated degree and metastasis. The expression in non-small cell lung cancer （NSCLC） was higher than that in small cell lung cancer （SCLC）; in well-moderately differentiated group was higher than that in poorly differentiated group; Earlier period group （I＋II） was higher than in later period group （Ⅲ＋Ⅳ）; and in group without lymphoid metastasis was higher than in patients with lymphoid metastasis. Conclusion： The progression of the lung cancer maybe related with the descended MRP-1/Cd9 expression, which may be useful in evaluating the prognosis of cancer patients.

Distribution characteristics of ammonia-oxidizing bacteria in the Typha latifolia constructed wetlands using fluorescent in situ hybridization(FISH)

A molecular biology method, fluorescent in situ hybridization（FISH）, in which the pre-treatment was improved in allusion to the media of the constructed wetlands（CW）, e.g. the soil and the grit, was used to investigate the vertical distribution characteristics of ammonia-oxidizing bacteria（AOB） quantity and the relation with oxidation-reduction potential（ORP） in the Typha latifolia constructed wetlands under three different Ioadings in summer from May to September. Results showed that the quantity of the AOB decreased in the Typha latifolia CW with the increase of vertical depth. However, the AOB quantity was 2-4 times the quantity of the control in the root area. Additionally, ORP in the rhizosphere was found to be higher than other areas, which showed that Typha latifolia CW was in an aerobic state in summer when using simulated non-point sewage at the rural area of Taihu Lake in China and small town combined sewage.