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Molecular subtyping of breast cancer intrinsic taxonomy with oligonucleotide microarray and NanoString nCounter

BIOSCIENCE REPORTS 2021年第8期41卷 BSR20211428页

作者： Chen, Yen-Jen Huang, Ching-Shui Phan, Nam-Nhut Lu, Tzu-Pin Liu, Chih-Yi Huang, Chi-Jung Chiu, Jen-Hwey Tseng, Ling-Ming Huang, Chi-Cheng Taipei Vet Gen Hosp Dept Surg Comprehens Breast Hlth Ctr Taipei Taiwan Cathay Gen Hosp Dept Surg Div Gen Surg Taipei Taiwan Taipei Med Univ Coll Med Sch Med Taipei Taiwan Acad Sinica Inst Informat Sci Taiwan Int Grad Program Bioinformat Program Taipei Taiwan Natl Taiwan Univ Grad Inst Biomed Elect & Bioinformat Taipei Taiwan Natl Taiwan Univ Inst Epidemiol & Prevent Med Taipei Taiwan Cathay Gen Hosp SiJhih Dept Pathol New Taipei Taiwan Cathay Gen Hosp Dept Med Res Taipei Taiwan Natl Def Med Ctr Dept Biochem Taipei Taiwan Natl Yang Ming Chiao Tung Univ Sch Med Inst Tradit Med Taipei Taiwan Natl Yang Ming Chiao Tung Univ Coll Med Sch Med Taipei Taiwan

Breast cancer intrinsic subtypes have been identified based on the transcription of a pre-defined gene expression (GE) profiles and algorithm (prediction analysis of microarray 50 gene set, PAM50). The present study compared molecular subtyping with oligonucleotide microarray and NanoString nCounter assay. A total of 109 Taiwanese breast cancers (24 with adjacent normal breast tissues) were assayed with Affymetrix Human Genome 0133 plus 2.0 microarrays and 144 were assayed with the NanoString nCounter while 64 patients were assayed for both platforms. Subtyping with the nearest centroid (single sample prediction (SSP)) was performed, and 16 out of 24 (67%) matched normal breasts were categorized as the normal breast-like subtype. For 64 breast cancers assayed for both platforms, 41 (65%, one unclassified by microarray) were predicted with an identical subtype, resulting in a fair K statistic of 0.60. Taking nCounter subtyping as the gold standard, prediction accuracy was 43% (3/7), 81% (13/16), 25% (5/20), and 100% (20/20) for basal-like, human epidermal growth factor receptor II (HER2)-enriched, luminal A and luminal B subtypes predicted from microarray GE profiles. microarray identified more luminal B cases from luminal A subtype predicted by nCounter. It is not uncommon to use microarray for breast cancer molecular subtyping for research. Our study showed that fundamental discrepancy existed between distinct GE assays, and cross-platform equivalence should be carefully appraised when molecular subtyping was conducted with oligonucleotide microarray.

关键词： breast cancer intrinsic subtype molecular subtyping NanoString nCounter oligonucleotide microarray PAM50

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Development of oligonucleotide microarray for accurate and simultaneous detection of avian respiratory viral diseases

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BMC VETERINARY RESEARCH 2019年第1期15卷 253-253页

作者： Xiao, Qian Yan, Liping Yao, Lu Lei, Jing Bi, Zhenwei Hu, Jianhua Chen, Yuqing Fang, An Li, Hui Li, Yuan Yan, Yan Zhou, Jiyong Nanjing Agr Univ Inst Immunol MOE Joint Int Res Lab Anim Hlth & Food Safety Nanjing 210095 Jiangsu Peoples R China Nanjing Agr Univ Inst Immunol Jiangsu Engn Lab Anim Immunol Nanjing 210095 Jiangsu Peoples R China Nanjing Agr Univ Coll Vet Med Jiangsu Detect Ctr Terr Wildlife Dis Nanjing 210095 Jiangsu Peoples R China Zhejiang Univ Key Lab Anim Virol Minist Agr Hangzhou 310058 Zhejiang Peoples R China Zhejiang Univ Affiliated Hosp 1 Collaborat Innovat Ctr Diag & Treatment Infect Di Hangzhou 310058 Zhejiang Peoples R China

Background: Avian influenza virus (AIV), infectious bronchitis virus (IBV), and Newcastle disease virus (NDV) are important avian pathogens that can cause enormous economic loss on the poultry industry. Different respiratory etiological agents may induce similar clinical signs that make differential diagnosis difficult. Importantly, AIV brings about severe threat to human public health. Therefore, a novel method that can distinguish these viruses quickly and simultaneously is urgently needed. Results: In this study, an oligonucleotide microarray system was developed. AIV, including H5, H7, and H9 subtypes;NDV;and IBV were simultaneously detected and differentiated on a microarray. Three probes specific for AIV, NDV, and IBV, as well as three other probes for differentiating H5, H7, and H9 of AIV, were first designed and jet-printed to predetermined locations of initiator-integrated poly(dimethylsiloxane) for the synchronous detection of the six pathogens. The marked multiplex reverse transcription polymerase chain reaction (PCR) products were hybridized with the specific probes, and the results of hybridization were read directly with the naked eyes. No cross-reaction was observed with 10 other subtypes of AIV and infectious bursal disease virus, indicating that the oligonucleotide microarray assay was highly specific. The sensitivity of the method was at least 100 times higher than that of the conventional PCR, and the detection limit of NDV, AIV, H5, H7, and H9 can reach 0.1 EID50 (50% egg infective dose), except that of IBV, which was 1 EID50 per reaction. In the validation of 93 field samples, AIV, IBV, and NDV were detected in 53 (56.99%) samples by oligonucleotide microarray and virus isolation and in 50 (53.76%) samples by conventional PCR. Conclusions: We have successfully developed an approach to differentiate AIV, NDV, IBV, H5, H7, and H9 subtypes of AIV using oligonucleotide microarray. The microarray is an accurate, high-throughput, and relatively simple

关键词： Avian influenza virus Newcastle disease virus Infectious bronchitis virus oligonucleotide microarray

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HIGHLY SENSITIVE H7N9 AVIAN INFLUENZA VIRUS DETECTION USING REVERSE TRANSCRIPTION LOOP-MEDIATED ISOTHERMAL AMPLIFICATION AND oligonucleotide microarray

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Taiwan Veterinary Journal 2016年第4期41卷 251-255页

作者： Lih-Chiann Wang Dean Huang School of Veterinary Medicine National Taiwan University No 1 Sec 4 Roosevelt Rd Taipei 10617 Taiwan

H7N9 avian influenza viruses have circulated in the human population and poultry flocks in China since 2013. H7N9 virus monitoring is imperative in Taiwan due to the frequent contact between China and Taiwan. Traditional viral molecular detection methods using RT-PCR and sequencing are time- and labor-intensive, thus a simpler and cheaper method with high sensitivity and specificity is worth developing. We successfully detected human and wild bird H7N9 viruses in this study using reverse transcription loop-mediated isothermal amplification (RT-LAMP) and oligonucleotide microarray. The detection limit was as low as one viral copy number. The specific matching reaction between the templates and microarray probes during hybridization ensured the high detection effectiveness. The excellent sensitivity and specificity of the RT-LAMP-microarray makes it a powerful H7N9 surveillance approach in Taiwan.

关键词： Avian influenza viruses H7N9 oligonucleotide microarray Reverse transcription loop-mediated isothermal amplification

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H5 avian influenza virus pathotyping using oligonucleotide microarray

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JOURNAL OF VIROLOGICAL METHODS 2015年 220卷 39-42页

作者： Wang, Lih-Chiann Huang, Dean Cheng, Ming-Chu Lee, Shu-Hwae Wang, Ching-Ho Natl Taiwan Univ Sch Vet Med Taipei 10617 Taiwan Council Agr Anim Hlth Res Inst Taipei Taiwan

The H5 avian influenza virus subtype has huge impact on the poultry industry. Rapid diagnosis and accurate identification of the highly pathogenic avian influenza virus and low-pathogenicity avian influenza virus is essential, especially during H5 outbreaks and surveillance. To this end, a novel and rapid strategy for H5 virus molecular pathotyping is presented. The specific hemagglutinin gene of the H5 virus and the basic amino acid number of the motif at the hemagglutinin precursor protein cleavage site were detected using oligonucleotide microarray. Highly pathogenic and low-pathogenicity avian influenza viruses in Taiwan were differentiated using 13 microarray probes with the naked eye. The detection limit reached 3.4 viral RNA copies, 1000 times more sensitive than reverse transcription polymerase chain reaction. Thus, the oligonucleotide microarray would provide an alternative H5 pathogenicity determination using the naked eye for laboratories lacking facilities. (C) 2015 Elsevier B.V. All rights reserved.

关键词： Avian influenza virus oligonucleotide microarray Pathotyping

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Avian oncogenic virus differential diagnosis in chickens using oligonucleotide microarray

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JOURNAL OF VIROLOGICAL METHODS 2014年 210卷 45-50页

作者： Wang, Lih-Chiann Huang, Dean Pu, Chang-En Wang, Ching-Ho Natl Taiwan Univ Sch Vet Med Taipei 10617 Taiwan Minist Justice Invest Bur Sci & Tech Res Ctr New Taipei City 23149 Taiwan

Avian oncogenic viruses include the avian leukosis virus (ALV), reticuloendotheliosis virus (REV) and Marek's disease virus (MDV). Multiple oncogenic viral infections are frequently seen, with even Marek's disease vaccines reported to be contaminated with ALV and REV. The gross lesions caused by avian oncogenic viruses often overlap, making differentiation diagnosis based on histopathology difficult. The objective of this study is to develop a rapid approach to simultaneously differentiate, subgroup and patho-type the avian oncogenic viruses. The oligonucleotide microarray was employed in this study. Particular DNA sequences were recognized using specific hybridization between the DNA target and probe on the microarray, followed with colorimetric development through enzyme reaction. With 10 designed probes, ALV-A, ALV-E, ALV-J, REV, MDV pathogenic and vaccine strains were clearly discriminated on the microarray with the naked eyes. The detection limit was 27 copy numbers, which was 10-100 times lower than multiplex PCR. Of 102 field samples screened using the oligonucleotide microarray, 32 samples were positive for ALV-E, 17 samples were positive for ALV-J, 6 samples were positive for REV, 4 samples were positive for MDV, 7 samples were positive for both ALV-A and ALV-E, 5 samples were positive for ALVA, ALV-E and ALV-J, one sample was positive for both ALV-J and MDV, and 3 samples were positive for both REV and MDV. The oligonucleotide microarray, an easy-to-use, high-specificity, high-sensitivity and extendable assay, presents a potent technique for rapid differential diagnosis of avian oncogenic viruses and the detection of multiple avian oncogenic viral infections under field conditions. (C) 2014 Elsevier B.V. All rights reserved.

关键词： Avian oncogenic viruses Avian leukosis virus Marek's disease virus oligonucleotide microarray Reticuloendotheliosis virus

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Response to drought and salt stress in leaves of poplar (Populus alba x Populus glandulosa): Expression profiling by oligonucleotide microarray analysis

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PLANT PHYSIOLOGY AND BIOCHEMISTRY 2014年 84卷 158-168页

作者： Yoon, Seo-Kyung Park, Eung-Jun Choi, Young-Im Bae, Eun-Kyung Kim, Joon-Hyeok Park, So-Young Kang, Kyu-Suk Lee, Hyoshin Korea Forest Res Inst Dept Forest Genet Resources Suwon 441847 South Korea Chungbuk Natl Univ Dept Hort Sci Cheongju 361763 South Korea Seoul Natl Univ Dept Forest Sci Seoul 151742 South Korea

Drought and salt stresses are major environmental constraints on forest productivity. To identify genes responsible for stress tolerance, we conducted a genome-wide analysis in poplar (Populus alba x Populus glandulosa) leaves exposed to drought and salt (NaCl) stresses. We investigated gene expression at the mRNA level using oligonucleotide microarrays containing 44,718 genes from Populus trichocarpa. A total of 1604 and 1042 genes were up-regulated (>= 2-fold;P value < 0.05) by drought and salt stresses, respectively, and 765 genes were up-regulated by both stresses. In addition, 2742 and 1685 genes were down-regulated by drought and salt stresses, respectively, and 1564 genes were down-regulated by both stresses. The large number of genes regulated by both stresses suggests that crosstalk occurs between the drought and salt stress responses. Most up-regulated genes were involved in functions such as subcellular localization, signal transduction, metabolism, and transcription. Among the up-regulated genes, we identified 47 signaling proteins, 65 transcription factors, and 43 abiotic stress-related genes. Several genes were modulated by only one of the two stresses. About 25% of the genes significantly regulated by these stresses are of unknown function, suggesting that poplar may provide an opportunity to discover novel stress-related genes. (C) 2014 Elsevier Masson SAS. All rights reserved.

关键词： Drought stress Gene expression oligonucleotide microarray Populus Salt stress

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

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JOURNAL OF BIOSCIENCES 2014年第5期39卷 795-804页

作者： Yan, Jing Yuan, Ying Mu, Runqing Shang, Hong Guan, Yifu China Med Univ Affiliated Hosp 1 Key Lab Med Cell Biol Minist EducDept Biochem & Mol Biol Shenyang 110001 Peoples R China China Med Univ Affiliated Hosp 1 Dept Lab Med Shenyang 110001 Peoples R China

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.

关键词： Bacillus isothermal probe locked nucleic acid oligonucleotide microarray

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Establishment and preliminary application of oligonucleotide microarray assay for detection of food-borne toxigenic microorganisms

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EUROPEAN FOOD RESEARCH AND TECHNOLOGY 2013年第6期236卷 1073-1083页

作者： Feng, Jiawang Wang, Xiaoyu Cao, Guiyun Hu, Songnan Kuang, Xiaoshan Tang, Shiming You, Shuzhu Liu, Lideng Zhuhai Entry Exit Inspect & Quarantine Bur Zhuhai 519015 Peoples R China Zhuhai Water Grp Co Ltd Water Qual Res Ctr Zhuhai 519000 Peoples R China

Rapid, high-throughput and accurate detection and identification of food-borne toxigenic microorganisms is crucial for food safety nowadays. An oligonucleotide microarray was designed and established and was applied to detect common food-borne toxigenic microorganisms in this study. PCR amplification of marker genes and 16S rRNA gene of 14 toxigenic bacteria and fungi using specific primers and oligo probes residing in these genes were employed and designed to fabricate the microarray. Optimization of hybridization conditions was implemented. The optimal conditions for hybridization were 51 A degrees C for 30 min. Furthermore, the ratio of biotin labeled to unlabeled primer for PCR amplification was also optimized to enhance specific hybridization of the microarray. Specificity, sensitivity (710 CFU/mL), and reproductivity assessment confirmed the practicability of the microarray. Finally, this microarray was successfully applied to detect 6 common toxigenic microorganisms from 328 food samples. The established microarray may provide potential for rapid detection and identification of toxigenic microorganisms from foods.

关键词： Toxigenic microorganism oligonucleotide microarray Food-borne Detection Optimization

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Development of a gold nanoparticle-based oligonucleotide microarray for simultaneous detection of seven swine viruses

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JOURNAL OF VIROLOGICAL METHODS 2013年第1期191卷 9-15页

作者： Wang, Xiaoqiang Dang, Erle Gao, Jinzhuai Guo, Sen Li, Zheng NW Univ Xian Coll Life Sci Xian 710069 Peoples R China

A gold nanoparticle (GNP) based oligonucleotide microarray assay (GNMA) that combined GNP-based multiplex asymmetric PCR with silver enhancement detection, was developed for simultaneous detection of seven important swine viruses in intensive swine production. Multiplex PCR was first performed to enable the target fragments of seven viruses containing a universal sequence, which were labeled simultaneously with GNPs by multiplex asymmetric PCR in the presence of excess GNP-conjugated primer. Target labeled products were captured by virus-specific oligonucleotide probes immobilized on microarrays, followed by silver staining for signal enhancement. Black image of microarray spots was easily detected by the naked eye or a simple flatbed scanner and quantified. The results for purified plasmid constructs indicated that the assay was highly specific for detecting the seven viruses in single or multiple infections, and as few as 6-80 copies/mu l of specific viral target fragments were detected successfully. Fifty-seven archived samples were tested by this assay, and the results were 100% consistent with previous results based on real-time PCR and those obtained by conventional PCR/RT-PCR and sequencing. The assay is appropriate for the routine diagnosis of viral infections in pigs due to its simplicity, low-cost, high specificity and sensitivity. (C) 2013 Elsevier B.V. All rights reserved.

关键词： Swine viruses oligonucleotide microarray Gold nanoparticles Multiplex asymmetric PCR

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Sex determination of bovine preimplantation embryos by oligonucleotide microarray

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ANIMAL REPRODUCTION SCIENCE 2013年第1-4期139卷 18-24页

作者： Yang, Hua Zhong, Fagang Yang, Yonglin Wang, Xinhua Liu, Shouren Zhu, Bin Xinjiang Acad Agr & Reclamat Sci Breed & Biotechnol Key Lab Sheep Bingtuan Shihezi 832000 Peoples R China Shanghai Baio Sci & Technol Ltd Co Shanghai 200233 Peoples R China

The aim has been to set up a rapid and accurate microarray assay using sandwich mode for sex determination of bovine preimplantation embryos. Twelve sequence-specific oligonucleotide capture probes used to discriminate 12 samples were spotted onto the aldehyde-modified glass slides by Arrayer. The 2 recognition probes used to identify coding regions of the sex-determining region of the Y chromosome gene (SRY) and beta-casein (CSN2) reference gene were coupled with biotin. The assay was optimized by using genomic DNA extracted from blood samples of known sex individuals. Polymerase chain reaction (PCR) was used to amplify the fragments in the HMG box region of SRY gene and CSN2 gene with sequence-specific primers. The sex of samples was identified by detecting both the SRY and CSN2 genes simultaneously in 2 reaction cells of microarrays, with the male having SRY and CSN2 signals and the female only CSN2. The sex of 20 bovine preimplantation embryos was determined by oligonucleotide microarray. The protocol was run with a blind test that showed a 100% (82/82) specificity and accuracy in sexing of leukocytes. The bovine embryos were transferred into 20 bovine recipients, with a pregnant rate of 40% (8/20). Three calves were born at term, and 5 fetuses were miscarried. Their sexes were fully in accordance with the embryonic sex predetermination predicted by oligonucleotide microarray. This suggests that the oligonucleotide microarray method of SRY gene analysis can be used in early sex prediction of bovine embryos in breeding programs. (c) 2013 Elsevier B.V. All rights reserved.

关键词： oligonucleotide microarray Sex determination SRY gene Bovine embryos

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