Development of an oligonucleotide microarray for simultaneous detection of two canine MDR1 genotypes and association between genotypes and chemotherapy side effects

Canine MDR1 gene mutations produce translated P-glycoprotein, an active drug efflux transporter, resulting in dysfunction or over-expression. The 4-base deletion at exon 4 of MDR1 at nucleotide position 230 (nt230[del4]) in exon 4 makes P-glycoprotein lose function, leading to drug accumulation and toxicity. The G allele of the c.-6-180T>G variation in intron 1 of MDR1 (single nucleotide polymorphism [SNP] 180) causes P-glycoprotein over-expression, making epileptic dogs resistant to phenobarbital treatment. Both of these mutations are reported to be common in collies. This study develops a more efficient method to detect these two mutations simultaneously, and clarifies the genotype association with the side effects of chemotherapy. Genotype distribution in Taiwan was also investigated. An oligonucleotide microarray was successfully developed for the detection of both genotypes and was applied to clinical samples. No 4-base deletion mutant allele was detected in dogs in Taiwan. However, the G allele variation of SNP 180 was spread across all dog breeds, not only in collies. The chemotherapy adverse effect percentages of the SNP 180 T/T, T/G, and G/G genotypes were 16.7%, 6.3%, and 0%, respectively. This study describes an efficient way for MDR1 gene mutation detection, clarifying genotype distribution, and the association with chemotherapy.

LNA-modified isothermal oligonucleotide microarray for differentiating bacilli of similar origin.

Oligonucleotide microarray has been one of the most powerful tools in the ‘Post-Genome Era’ for its high sensitivity, high throughput and parallel processing capability. To achieve high detection specificity, we fabricated an isothermal microarray using locked nucleic acid (LNA)-modified oligonucleotide probes, since LNA has demonstrated the advanced ability to enhance the binding affinity toward their complementary nucleotides. After designing the nucleotide sequences of these oligonucleotide probes for gram-positive bacilli of similar origin (Bacillus subtilis, Bacillus licheniformis, Bacillus pumilus, Bacillus megaterium and Bacillus circulans), we unified the melting temperatures of these oligonucleotide probes by modifying some nucleotides using LNA. Furthermore, we optimized the experimental procedures of hydrating microarray slides, blocking side surface as well as labelling the PCR products. Experimental results revealed that KOD Dash DNA polymerase could efficiently incorporate Cy3-dCTP into the PCR products, and the LNA-isothermal oligonucleotide microarray were able to distinguish the bacilli of similar origin with a high degree of accuracy and specificity under the optimized experimental condition.

Design of 16 S rRNA-based Oligonucleotide Array Using Group-specific Non-unique Probes in Large Scale Bacteria Detection / 生物化学与生物物理进展

With thousands of sequenced 16 S rRNA genes available,and advancements in oligonucleotide microarray technology,the detection of microorganisms in microbial communities consisting of hundreds of species may be possible.The existing algorithms developed for sequence-specific probe design are not suitable for applications in large-scale bacteria detection due to the lack of coverage,flexibility and efficiency.Many other strategies developed for group-specific probe design focus on how to find a unique group-specific probe that can specifically detect all target sequences of a group.Unique group-specific probe for each group can not always be found.Hence,it is necessary to design non-unique probes.Each probe can specifically detect target sequences of a different subgroup.Combination of multiple probes can achieve higher coverage.However,it is a time-consuming task to evaluate all possible combinations.A feasible algorithm using relative entropy and genetic algorithm (GA) to design group-specific non-unique probes was presented.

Transcriptional profiling and Wnt signaling activation in proliferation of human hepatic stellate cells induced by PDGF-BB / 대한간학회지

BACKGROUND/AIMS: This study aimed to better understand gene expression profiles of human hepatic stellate cell (HSC) activation and the relationship with the Wnt signaling pathway. METHODS: The global transcript levels in platelet derived growth factor-BB (PDGF-BB)-stimulated hTERT HSCs were analyzed using oligonucleotide microarrays. Oligonucleotide microarrays with 19K human oligo chips were performed to obtain gene expression profiles associated with proliferation in human hTERT HSCs. The microarray data was verified by real time quantitative PCR and expression of the components of Wnt signaling was analyzed by Western blot. RESULTS: Microarray data showed 243 up-regulated and 265 down-regulated genes in PDGF-BB-treated HSCs. The changes in expression of glypican3 and BH3 interacting domain death agonist (BID) mRNA in real time quantitative PCR, especially among the highly up- or down-regulated genes, were statistically consistent with the microarray data. The Wnt signaling pathway components, frizzled10 (FZD10) and calcium/calmodulin-dependent protein kinase II alpha (CAMK2A), showed increased expression in the short time course microarray and the up-regulation of FZD10 also occurred at the protein level. Our data showed various gene expression profiles during activation of human HSC. CONCLUSIONS: The up-regulated expression of FZD10 and CAMK2A suggests that the Wnt/Ca2+ signaling pathway is active in hTERT HSCs and may participate in HSC activation and proliferation

Construction and preliminary application of oligonucleotide microarray specialized for pancreatic adenocarcinoma / 中国病理生理杂志

AIM:To investigate the construction of oligonucleotide microarray specialized for pancreatic adenocarcinoma-associated genes and its preliminary application of detecting differential expressed genes in pancreatic cancer.METHODS:Pancreatic cancer related genes were purposely selected,and oligonucleotide microarray was prepared by spotting oligonucleotide probes on glass slides coated with APS-PDC.Labeled cDNA targets for hybridizations were synthesized by reverse transcription from total RNA in the presence of Cy5-dCTP and Cy3-dCTP,respectively.Hybridized microarray was scanned by Agilent laser scanner,and the aquired image was analyzed by Imagene3.0 software.The intensity ratios of Cy3 and Cy5 were calculated.To confirm the expression profiles of these genes,quantitative reverse transcription-PCR (QRT-PCR) was carried out for CDC25B and TUSC3 genes,and β-actin gene was taken as internal control.The product of PCR was quantitated by comparative Ct method.RESULTS:The signal of microarray hybridization was clear,and the images had a lower background and higher signal-noise ratio.In comparison with normal pancreas,twenty -four differentially expressed genes were identified which included seventeen up-regulated and seven down-regulated genes.The results of QRT-PCR demonstrated that the expressions of CDC25B and TUSC3 in pancreatic cancer were up-regulated and down-regulated respectively,which is consistent with microarray results.CONCLUSION:The oligonucleotide microarray specialized for pancreatic cancer is desirable for its specialty and sensitivity,which can simultaneously and parallelly detect multiple pancreatic cancer-associated genes.

Clinical Efficacy of Human Papillomavirus DNA Detection in Urine from Patients with Various Cervical Lesions

A considerable number of adult Korean women avoid a Pap smear due to fear and discomfort of the pelvic examination. A reliable but noninvasive and comfortable screening method would considerably increase the participation rate. To evaluate the clinical efficacy of urine-based human papillomavirus (HPV) detection by oligonucleotide microarray, the results of HPV test from matched cervical swab specimens were compared. HPV DNA was detected in 70 of 100 cervical samples. HPV 16 was the most prevalent type (38/70), followed by types 18, 58, 52, 33, 35, 31, and 51. HPV DNA was identified in 47 of 90 urine samples. HPV 16 was the most prevalent type (30/45), followed by types 18, 52, 35, 51, 58, 33, and 56. The HPV detection rates of the cervical swabs increased in accordance with the severity of the cytologic and histologic diagnosis. The type specific agreement of HPV DNA tests between cervical swabs and urine was good in HPV 16 (kappa index=0.64 [95% CI: 0.50-0.79]), 18, 52, and 58 and fair in HPV 33 and 35. We propose that a urine HPV test is a valuable adjunctive method for a conventional Pap smear and can be used in population screening for cervical cancer in countries where it is difficult to obtain colposcopic specimens for cultural or religious reasons.

Differentially Expressed Cellular Gene Profiles between Healthy HIV-infected Koreans and AIDS Patients

BACKGROUND: The global effect of HIV infection on the host cell gene expression profiles in healthy HIV-infected patients, as long-term non-progressors, remains largely unknown. To identify the cellular genes related with HIV infection and delayed disease progression in vivo, the host gene expression profiles between healthy HIV-infected Koreans and AIDS patients were investigated. METHODS:Differential expression gene analysis was performed via oligonucleotide microarray with using Magic-oligo 10K chip. Ten HIV-uninfected persons and 10 HIV-infected patients (healthy HIV-infected patients vs. AIDS patients. respectively) were studied. RESULTS: Only 10.8% (1,097 genes) of the total genes, that is, 331 up-regulated genes and 766 down- regulated genes were differentially expressed with more than a two-fold change in the HIV-infected persons as compared to those of the HIV-uninfected persons. Especially, 97 genes (8.8%) among 1,097 genes were commonly up- or down-regulated in both the healthy HIV-infected patients and the AIDS patients. 187 genes were differently expressed on the gene expression analysis between the healthy HIV-infected patients and the AIDS patients. Twenty-eight genes out of them showed very significant differences with a P value <0.01. Especially, tripartite motif (TRIM) 14 protein and interferon gamma receptor 2 were dramatically up-regulated in healthy HIV-infected patients, while death-associated protein, DNA directed RNA polymerase II polypeptide A and STAT were over-expressed in AIDS patients. CONCLUSION: Although this microarray study has some limitations, the above results will be helpful for performing more detailed, future functional studies on the differentially expressed genes related to HIV infection and delayed disease progression in vivo.

Gene Expression Profiling using Oligonucleotide Microarray in Atrophic Gastritis and Intestinal Metaplasia / 대한소화기학회지

BACKGROUND/AIMS: The atrophic gastritis with intestinal metaplasia of gastric mucosa has been considered to be the major factor of carcinogenesis in the stomach. However, the key molecules are still poorly understood. To elucidate the molecular genetic basis, we report the results of our initial microarray data to analyze the genome pattern in patients with atrophic gastritis and intestinal metaplasia of the stomach. METHODS: We used oligonucleotide microarray technique to evaluate the gene expression profiles in atrophic gastritis with intestinal metaplasia, in comparison with those of normal mucosa. For the identification of differentially expressed genes, Significance Analysis of Microarrays (SAM) package method was used. The results were analyzed using global normalization, intensity dependent normalization, and box plot normalization. RESULTS: Eight genes including FABP, REG, OR6C1, MEP1, SLC6A1, SI, Mucin 1, and RAB23 in mucosa of atrophic gastritis and intestinal metaplasia were up-regulated by more than 10 times as compared with normal gastric mucosa. Only one gene, LOC44119 was down-regulated by more than 10 times of the expression as compared with normal gastric mucosa. In respect to the expression of known genes related to gastric carcinogenesis, 8 genes including FN1, SRMS, TP53, TP53IMP2, TP53I3, FGFR4, TGFB1, and TGFA showed up- and down-regulations more than 2 folds in expression pattern. CONCLUSIONS: We could identify a total genome pattern in patient with atrophic gastritis and intestinal metaplasia using oligonucleotide microarray. We believe that the current results will serve as a fundamental bioinformative basis for clinical applications in diagnosis and treatment of gastric cancer and precancerous lesion in the future.

Evaluation of Two Different Sample Labeling Methods on Background Signal Intensities for 60 mer Oligonucleotide Microarrays / 中国生物工程杂志

The effects of two different sample labeling methods on background signal intensities for high-density 60mer oligonucleotide microarray were investigated. Peripheral blood samples from five disease and five control subjects were collected. Total RNA targets from peripheral blood mononuclear cells were extracted and labeled with RD-PCR protocol, which were hybridized to Agilent Human 1B oligonucleotide microarrays in a two-color comparative format. The positive control targets were labeled with the directly incorporated fluorescently-labeled dNTP labeling. The SPSS program was performed to test normality of the dataset, variance homogeneity between the groups, coefficients of variation (CV) and analysis of variance. The results showed that the background signal intensities of Cy3 channel were higher than those of Cy5 channel. The differences of background signal intensities between the RD-PCR approach and the directly incorporated fluorescently-labeled dNTP labeling were extremely significant (P- Cy3

When should human papillomavirus (HPV) testing be done after conization? / 대한산부인과학회지

OBJECTIVE: To know when human papillomavirus (HPV) testing should be done after conization. METHODS: Between 1997 to 2004, Large Loop Excisions of the Transformation Zone (LLETZ) were done for conization to women with cervical pathology at A University Hospital. The Pap and HPV typing were done before LLETZ procedures. After conizations, HPV typing were planned to be done every 3 months. Every HPV typing was done by HPV oligonucleotide microarray (Biomedlab Co., Seoul, South Korea). RESULTS: For 8 years, 120 LLETZ were enrolled in this study. There were 8 cases of no neoplasm, 9 cases of CIN 1, 17 cases of CIN 2, 74 cases of CIN 3, 10 cases of microinvasive cervix cancer, and 2 cases of adenocarcinoma in situ. HPV DNA before LLETZ procedures was found about 85.0% and subtype 16 was the most common type among the patients with cervical lesion (40.8%). After LLETZ, 190 HPV typing were done through 1,307 total months (average, 6.9 months/typing). 95 (79.2%) cases had negative results, and 25 (20.8%) cases had positive results. Our data showed that, after conization, about 80% turned out to negative in 6 months. CONCLUSION: Our data suggested HPV DNA testing should be done after 6 months of LLETZ, as about 80% were destined to negative in 6 months.

Multiply Labeled Primers Amplifying Fluorescent Signal on Oligonucleotide Microarray / 生物化学与生物物理进展

Oligonucleotide microarray technology is a powerful data-mining platform and has been widely applied in biosciences. To improve the performance of assays on the oligonucleotide microarray, the factors that influence the hybridization effects such as surface chemistry, probe size, spacer length, hybridization conditions etc were intensely studied and optimized. However, it is a key problem with DNA microarrays how to generate higher fluorescent signals to improve the detection sensitivity. Two types of multiply labeled primers, termed multiply labeled linear primer and multiply labeled branched primer, were used to enhance the fluorescent signal obtained from two-dimensional DNA microarrays.The signal was intensified by increasing the number of fluorophores labeled on the target DNA segment. It was indicated that the detection limit (minimum template amoumt for detection) of the multiply labeled primers is about 1% of that of the singly labeled primer. Multiple labeling is an effective signal amplification method to increase the detection sensitivity of the probes in a miniaturized array format.

Detection of Mycoplasma Infection in Cultured Cells on the Basis of Molecular Profiling of Host Responses

Adaptive responses to diverse microbial pathogens might be limited in relatively few types. Host cell responses to pathogens are believed to be patterned or stereotyped along with species or class. We tried to compose the host response to Mycoplasma in terms of cellular gene expression. Although gene expression profile of two host HeLa and 293 cells were quite different each other, 30 genes were differentially expressed by mycoplasma infection in both of HeLa and 293 cells. Six of them (PR48, MADH4, MKPX, CRK, RBM7, NEK3) were related to cell cycle or proliferation. Another category of genes like IL1HY1, KLRF1, TNFSF14, GBP1 were host defense to elicit immune responses. With this set of genes, we establish the prediction model for mycoplasma contamination.

The Report of the Results of HPV Oligonucleotide Microarray Tested on the First Voided Urine of Patients of CIN and Cervix Cancer / 대한산부인과학회잡지

OBJECTIVE: To know whether HPV Oligonucleotide Microarray (HPVDNAChip) can detect the HPV DNA in the urine and, if it can, to compare the results with Pap smear, biopsy, and cervix HPVDNAChip. METHODS: The authors had done Pap smear, cervix HPVDNAChip and colposcopy-guided punch biopsy as well as detailed information to those who visited Dept. of Ob. And Gyn. during 1st of April to 31st of May in 2003 for their uterine cervical problems related to the neoplasia. When they were determined to admit for treatment, urine had been collected to be tested by HPVDNAChip. RESULTS: Among 25 patients enrolled in this study, there were 10 whose urine HPVDNAChip test turned out positive (40%). Among 10 positive results, 9 patients had HPV 16 subtypes. Among 10 urine HPVDNAChip positive patients, there were 5 HSIL, 4 squamous cell cancer (SCC), and 1 ASCUS cell types on the Pap smears. Among 15 urine HPVDNAChip negative patients, there were 7 HSIL, 5 SCC, 1 ASCUS, 1 LSIL, and 1 AGUS. Among 10 urine HPVDNAChip there are 5 CIN3, and 4 invasive SCC, and 1 adenocarcinoma at the biopsy. Among 15 urine HPVDNAChip negative patients, there are 7 CIN3, 6 invasive SCC, 1 adenocarcinoma in situ, and 1 CIN1 patient. Whenever there were a urine HPVDNAChip 16 subtype positive, there were always cervix HPVDNAChip 16 subtype positive, but among the 12 urine HPVDNAChip negative patients, 5 had HPV 16 subtype positive and 4 had another subtypes and 3 had negative on cervix HPVDNAChip tests. CONCLUSION: Using HPVDNAChip, we verified that 40% of patients had the HPV DNA in their urine who had admitted for the treatment of their cervical neoplasm. And HPV 16 subtype was the most common type in the urine. If we can extend this data more widely, we might use it as an auxiliary tool for cervical HPV infection.

Differential expressions of mitochondrial genome and apoptosis related genes in A549 cell line induced by cisplatin / 解放军医学杂志

Objective To investigate the differential expression of mitochondrial genome and apoptosis-related genes in human lung adenocarcinoma A549 cell line induced by cisplatin. Methods A549 cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS; Hyclone) in 150-mm dishes, and the test cells were exposed to 0.5?g/ml cisplatin continuously for 20h. Same volume PBS solution was added to culture of control cells instead of cisplatin. Human mitochondrial genome oligonucletide micoarray was used to detect the differential expression of 26 target genes. The apoptosis status was analyzed by the flow cytometry technique. Results Compared with the control cells, the expression levels of tRNA-Cys, tRNA-Asn, 12S rRNA and cytochrome oxidase subunit I were remarkably elevated in test cells. The expression levels of bax, NADH dehydrogenase subunit I, NADH dehydrogenase subunit Ⅱ and tRNA-Ser, tRNA-Asp, tRNA-Arg and tRNA-Ala also were elevated to some extent. Apoptosis rate of test cells was 8.5%?0.78%(n=5), which was remarkably higher than that of control cells 1.3%?0.56%(n=5,P

Comparison of CYP2D6 genotyping by allele-specific PCR with DXT phenotype and gene chip testing / 中国临床药理学与治疗学

AIM: To evaluate association of genotype and phenotype of CYP2D6 and the application of oligonucleotide microarray hybridization genetic testing in CYP2D6 multiple alleles analyses. METHODS: Two hundred forty-two healthy volunteers were recruited, and a 60 mg oral dose of dextromethorphan (DXT) was administered to each for assessment of the DXT metabolic ratio . A 20 ml blood sample was also collected for DNA isolation and testing. CYP2D6 alleles *2--*11; *17 and multiple CYP2D6 gene copies were tested by allele-specific PCR and/or the affymetrix CYP450 gene chip assay. Five of the CYP2D6 alleles (*3, *4, *6, *7, and *9) were evaluated by both PCR and the CYP450 gene chip assay. RESULTS: The CYP2D6 genotype was more informative and reflective in CYP2D6 enzyme expression than a phenotype. Genetic tests for the CYP2D6 *3, *4, *6, *7 and *9 alleles showed a high degree of concordance between the CYP450 gene chip and AS-PCR methods. CONCLUSION: Oligonucleotide microarray hybridization is a promising and reliable approach for detecting multiple alleles at gene loci.

Effects of component combination in Siwu Decoction on proliferation of human bone marrow stromal cell lines HFCL and hematopoiesis-related gene expression / 中草药

Objective To investigate the effects of component combination in Siwu Decoction (CCSWD) on the proliferation of human bone marrow stromal cell lines HFCL and hematopoiesis-related gene expression and to probe into the mechanism of the stimulative effects of SWD and CCSWD on the proliferation of HFCL cells. Methods MTT and FCM were used for assaying the proliferation potency and cell cycle of HFCL cells, respectively. A human hematopoiesis-related cytokine oligonuleotide microarray was prepared to investigate the diversity of gene expression of HFCL cells in SWD and CCSWD groups. Results The energy metabolism and proliferation potency of HFCL cells treated with CCSWD were more prompted than that of the control group. Meanwhile, the percentage of HFCL cells in G 0/G 1 phase treated with CCSWD was significantly lower, while the proliferation index (PI) was higher than that of the control group. Compared to the control group, expressions of IL 2-R?, NF-?B, and TPO were up-regulated in CCSWD group.Conclusion The proliferation of HFCL cells could be facilitated by CCSWD with up-regulating the expressions of IL 2-R?, NF-?B, and TPO.

Studies with an oligonucleotide microarray on the changes of gene expression related with NB4 apoptosis induced by arsenic trioxide / 解放军医学杂志

Objective Gene microarray technology was used to investigate the differential gene expression of apoptosis related genes in NB4 cells induced by arsenic trioxide. Methods The databases of Evntrez and Human IPI were searched with "apoptosis or apoptotic" as the key words, and 1384 apoptosis related genes were found after the redundant genes were eliminated by chromosomal localization. The probes of these genes were designed using OligoArray 2.0, and then analyzed by BLAST. All the probes were immobilized on the glass slide, which were used as oligonucleotide microarray. After NB4 cells were treated with 2umol/L As2O3 for 48h, the total RNA were extracted. cDNAs of control group and test group were fluorescently labeled with Cy3 and Cy5, respectively, by RT-PCR. The fluorescent samples were hybridized with an oligonucleotide microarray containing 1384 apoptosis related genes to search for the differentially expressed genes in the cells with or without As2O3 treatment. The hybridization signals were scanned by oligonucleotide microarray, and then the fluorescent intensity of Cy3 and Cy5 and the ratio of two fluoresceins were analyzed using certain software. Then the differential expressed genes were analyzed after As2O3 treatment, in which the most distinctly differential expressed genes were chosen as targets, and through PCR amplification and gel electrophoresis the above genes were verified. Results There are 4 genes up-regulated and 12 genes down-regulated in expression in NB4 cells after 48h treatment with 2umol/L As2O3, which were in accordance with the results of RT-PCR and oligonucleotide microarray. Conclusion Differential gene expression in NB4 cells was induced by As2O3 treatment. These differentially expressed genes, with relation to signal transduction, transcription regulation, cell cycles, oxidation response, protein translation and cell differentiation, may play an important role in NB4 cell apoptosis.