• Proceedings of the Korean Vacuum Society Conference
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• 2013.08a
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• pp.88-89
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• 2013

A variety of influenza A viruses from animal hosts are continuously prevalent throughout the world which cause human epidemics resulting millions of human infections and enormous industrial and economic damages. Thus, early diagnosis of such pathogen is of paramount importance for biomedical examination and public healthcare screening. To approach this issue, here we propose a fully integrated Rotary genetic analysis system, called Rotary Genetic Analyzer, for on-site detection of influenza A viruses with high speed. The Rotary Genetic Analyzer is made up of four parts including a disposable microchip, a servo motor for precise and high rate spinning of the chip, thermal blocks for temperature control, and a miniaturized optical fluorescence detector as shown Fig. 1. A thermal block made from duralumin is integrated with a film heater at the bottom and a resistance temperature detector (RTD) in the middle. For the efficient performance of RT-PCR, three thermal blocks are placed on the Rotary stage and the temperature of each block is corresponded to the thermal cycling, namely $95^{\circ}C$ (denature), $58^{\circ}C$ (annealing), and $72^{\circ}C$ (extension). Rotary RT-PCR was performed to amplify the target gene which was monitored by an optical fluorescent detector above the extension block. A disposable microdevice (10 cm diameter) consists of a solid-phase extraction based sample pretreatment unit, bead chamber, and 4 ${\mu}L$ of the PCR chamber as shown Fig. 2. The microchip is fabricated using a patterned polycarbonate (PC) sheet with 1 mm thickness and a PC film with 130 ${\mu}m$ thickness, which layers are thermally bonded at $138^{\circ}C$ using acetone vapour. Silicatreated microglass beads with 150~212 ${\mu}L$ diameter are introduced into the sample pretreatment chambers and held in place by weir structure for construction of solid-phase extraction system. Fig. 3 shows strobed images of sequential loading of three samples. Three samples were loaded into the reservoir simultaneously (Fig. 3A), then the influenza A H3N2 viral RNA sample was loaded at 5000 RPM for 10 sec (Fig. 3B). Washing buffer was followed at 5000 RPM for 5 min (Fig. 3C), and angular frequency was decreased to 100 RPM for siphon priming of PCR cocktail to the channel as shown in Figure 3D. Finally the PCR cocktail was loaded to the bead chamber at 2000 RPM for 10 sec, and then RPM was increased up to 5000 RPM for 1 min to obtain the as much as PCR cocktail containing the RNA template (Fig. 3E). In this system, the wastes from RNA samples and washing buffer were transported to the waste chamber, which is fully filled to the chamber with precise optimization. Then, the PCR cocktail was able to transport to the PCR chamber. Fig. 3F shows the final image of the sample pretreatment. PCR cocktail containing RNA template is successfully isolated from waste. To detect the influenza A H3N2 virus, the purified RNA with PCR cocktail in the PCR chamber was amplified by using performed the RNA capture on the proposed microdevice. The fluorescence images were described in Figure 4A at the 0, 40 cycles. The fluorescence signal (40 cycle) was drastically increased confirming the influenza A H3N2 virus. The real-time profiles were successfully obtained using the optical fluorescence detector as shown in Figure 4B. The Rotary PCR and off-chip PCR were compared with same amount of influenza A H3N2 virus. The Ct value of Rotary PCR was smaller than the off-chip PCR without contamination. The whole process of the sample pretreatment and RT-PCR could be accomplished in 30 min on the fully integrated Rotary Genetic Analyzer system. We have demonstrated a fully integrated and portable Rotary Genetic Analyzer for detection of the gene expression of influenza A virus, which has 'Sample-in-answer-out' capability including sample pretreatment, rotary amplification, and optical detection. Target gene amplification was real-time monitored using the integrated Rotary Genetic Analyzer system.

• Horticultural Science & Technology
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• v.32 no.1
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• pp.91-99
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• 2014

The objective of this study is to identify cold-tolerance genes in Brassica rapa. In order to acheive this goal, we analyzed a KBGP-24K oligo chip data [BrEMD (B. rapa EST and Microarray Database)] using B. rapa ssp. pekinensis inbred line 'Chiifu' under cold stress condition ($4^{\circ}C$). Among 23,929 unigenes of B. rapa, 417 genes (1.7%) were primarily identified as cold responsive genes that were expressed over 5-fold higher than those of wild type control, and then a gene which has unknown function and has full length sequence was selected. It was named BrCSR (B. rapa Cold Stress Resistance). BrCSR was transformed using expression vector pSL101 to confirm whether BrCSR can enhance cold tolerance in tobacco plants. $T_1$ transgenic tobacco plants expressing BrCSR were selected by PCR and Southern hybridization analyses, and the function of BrCSR was characterized by expression level analysis and phenotype observation under cold stress condition. The expression level of BrCSR in transgenic tobacco plants increased up to about two folds in quantitative real-time RT-PCR assay and this was very similar to Northern blot hybridization analysis. Analysis of phenotypic characteristics clearly elucidated that transgenic tobaccos expressing BrCSR were more cold tolerant than wild type control under $4^{\circ}C$ treatment. Based on these results, we conclude that the over-expression of BrCSR might be closely related to the enhancement of cold tolerance.

• Journal of Sasang Constitutional Medicine
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• v.11 no.2
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• pp.185-194
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• 1999

Recently some report has been published on the classification of Sasang constitution by analysis of genetic polymorphism. In this study, to present the ideas in the study on the classification of Sasang constitution based on the genetic variation of individuals, we reviewed the previous studies dealing with relationship between genetic polymorphism and Sasang constitution and suggested new molecular biological methodologies applicable to the classification of Sasang constitution. The results were as follow; 1. In the studies on the classification of constitution by analysis of genetic polymorphism, the special genes were desirable as subjects rather than the non-functional part of genome such as inter-gene and intron. Since microsatellite is usually located in inter-gene or intron, representation of constitutions may not associated with the polymorphism of certain microsatellite. 2. It may possible that polymorphism patterns of the genes related to the expression of individuality is associated with the types of Sasang constitutions. Therefore, angiotensin converting enzyme (ACE) which known have some role in the determination the human performance is one of the candidate genes can use for the classification of Sasang constitutions. 3. It is well known that HLA types are very variable in human and closely associated with the determination of susceptibility for certain disease. The studies on the polymorphism of HLA types may applicable to the classification of Sasang constitutions. 4. DNA chip technique which developed recently for the analysis of the expression of human genes may powerful tools in the study of Sasang medicine.

• Journal of Life Science
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• v.26 no.7
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• pp.789-795
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• 2016

A lymph node (LN) is one of the secondary lymphoid organs. An LN consists of a complicated 3 dimensional frame structure and several stromal cells. Fibroblastic reticular cells (FRC) are distributed in the T zone for interaction with T cells. FRC secrete homing chemokines such as CCL19 and CCL21. Moreover, FRC play a pivotal role in the production of extracellular matrix (ECM) into LN for ECM reorganization against pathogen infections. However, not much is known about the involvement of the immune reaction of FRC. The present report is for the characterization of FRC on immune response. For this, FRC were positioned in several infected situations such as co-culture with macrophage, lipopolysaccharide (LPS), and TNFα stimulation. When a co-culture between FRC and macrophage was performed, a morphological change in FRC was observed, and empty space between FRCs was created by this change. The soluble ICAM-1 protein level was up-regulated by co-culturing with Raw264.7 and the treatment of the ROCK inhibitor Y27632. The activity of matrix metalloproteinase (MMP) was up-regulated by LPS onto FRC. Furthermore, the inflammatory cytokine TNFα regulated the expression of ECM in FRC by a gene chip assay. Collectively, it suggests that FRC are involved in immune reactions.

• The Journal of Korean Academy of Prosthodontics
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• v.55 no.4
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• pp.361-371
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• 2017

Purpose: We aimed to investigate the gene expression of human gingival fibroblasts on microgroove surface using DNA microarray. Materials and methods: Microgrooves were applied on grade II titanium discs to have 0/$0{\mu}m$ (NE0, control group), 60/$10{\mu}m$ (E60/10, experimental group) of respective width/depth by photolithography. The entire surface of the microgrooved Ti substrata was further acid etched and used as the two experimental groups in this study. Human gingival fibroblasts were cultured in the experimental group and the control group, and total RNA was extracted. The oligonucleotide microarray was performed to confirm the changes of various gene expression levels between experimental group and control group. Changes of gene expression level were determined at the pathway level by mapping the expression results of DNA chips, using the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis. Results: Gene expression levels on E60/10 and NE0 were analyzed, there were 123 genes showing significant differences in expression more than 1.5 times on E60/10 microgrooved surface compared to NE0 surface, and 19 genes showing significant differences in expression more than 2 times. The KEGG pathway analysis confirmed the changes in gene expression levels under experimental conditions. Cell signaling, proliferation, and activity among the various gene expression results were identified. Conclusion: Microgrooved surfaces induce gene expression changes and related cell signaling. According to the results of this study, microgrooves can be used as the surface of various biomaterials which need to improve cell activity through gene expression changes and activation of cell signaling.

• Proceedings of the Korean Society of Applied Pharmacology
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• 2003.11a
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• pp.98-98
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• 2003

${\beta}$-(1,3)-D-Glucans have been known to exhibit antitumor and antimicrobial activities. The presence of dectin-1,${\alpha}$, ${\beta}$-glucan receptor of dendritic cell, on macrophage has been controvertial. RT-PCR analysis led to the detection of dectin-1${\alpha}$ and ${\beta}$ in murine macrophage Raw264.7 cell line. Among the various organs of mouse, dectin-1${\alpha}$ and ${\beta}$ were detected in the thymus, lung, spleen, stomach and intestine. To analyze gene expression modulated by ${\beta}$-glucan treated murine Raw264.7 macrophage, total mRNA was applied to cDNA microarray to interrogate the expression of 7,000 known genes. cDNA chip analysis showed that ${\beta}$-glucan of P. osteatus increased gene expressions of immunomodulating genes, membrane antigenic proteins, chemokine ligands, complements, cytokines, various kinases, lectin associated genes and oncogenes in Raw 264.7 cell line. When treated with ${\beta}$-glucan of P. osteatus and LPS, induction of gene expression of TNF-${\alpha}$ and IFN-R1 was confirmed by RT-PCR analysis. Induction of TNF-R type II expression was confirmed by FACS analysis. IL-6 expression was abolished by EDTA in ${\beta}$-glucan and LPS treated Raw264.7 cell line, indicating that ${\beta}$-glucan binds to dectin-l in a Ca$\^$＋＋/ -dependent manner. To increase antitumor efficacy of ${\beta}$-glucan, ginsenoside Rh2 (GRh2) was co-treated with ${\beta}$-glucan in vivo and in vitro tests. IC$\sub$50/ values of GRh2 were 20 and 25 $\mu\textrm{g}$/$m\ell$ in SNU-1 and B16 melanoma F10 cell line, respectively. Co-treatment with ${\beta}$-glucan and GRh2 showed synergistic antitumor activity with cisplatin and mitomycin C both in vitro and in vivo. Single or co-treatment with ${\beta}$-glucan and GRh2 increased tumor bearing mouse life span. Co-treatment with ${\beta}$-glucan and GRh2 showed more increased life span with mitomycin C than that with cisplatin. Antitumor activities were 67% and 72 % by co-injection with ${\beta}$-glucan and GRh2 in the absence or presence of mitomycin C, respectively.

• Proceedings of the Korean Society for Bioinformatics Conference
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• 2006.02a
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• pp.83-89
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• 2006

Cellular functions are carried out by a concerted action of biochemical pathways whose components have genetic interactions. Abnormalities in the activity of the genes that constitute or modulate these pathways frequently have oncogenic implications. Therefore, identifying the upstream regulatory genes for major biochemical pathways and defining their roles in carcinogenesis can have important consequences in establishing an effective target-oriented antitumor strategy We have analyzed the gene expression profiles of human liver cancer samples using cDNA microarray chips enriched in liver and/or stomach-expressed cDNA elements, and identified groups of genes that can tell tumors from non-tumors or normal liver, or classify tumors according to clinical parameters such as tumor grade, age, and inflammation grade. We also set up a high-throughput cell-based assay system (cell chip) that can monitor the activity of major biochemical pathways through a reporter assay. Then, we applied the cell chip platform for the analysis of the HCC-associated genes discovered from transcriptome profiling, and found a number of cancer marker genes having a potential of modulating the activity of cancer-related biochemical pathways such as E2F, TCF, p53, Stat, Smad, AP-1, c-Myc, HIF and NF-kB. Some of these marker genes were previously blown to modulate these pathways, while most of the others not. Upon a fast-track phenotype analysis, a subset of the genes showed increased colony forming abilities in soft agar and altered cell morphology or adherence characteristics in the presence of purified matrix proteins. We are currently analyzing these selected marker genes in more detail for their effects on various biological Processes and for Possible clinical roles in liver cancer development.

• Analytical Science and Technology
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• v.22 no.6
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• pp.498-503
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• 2009

Lung cancer is the main cancer on the world today, due to the high case fatality. Lung cancer can devide into two major types, such as small-cell lung cancer (SCLC) and non-small-cell lung cancer (NSCLC). Mutations in the epidermal growth factor receptor (EGFR) have been described in patients with advanced NSCLC. Mutations in the EGFR are associated with clinical and radiographic responses to EGFR tyrosine kinase inhibitors gefitinib and erlotinib. Thus, the detection of EGFR mutation can offer an effective information in clinical decision-making. In this study, We developed very simple, cheep and rapid mutation detection system by chip-based isothermal amplification method. The method described here has shown the advantages of rapid amplification, high sensitivity, and specificity. Also, it will be useful for rapid and reliable clinical diagnosis of EGFR mutation.

• Clinical and Experimental Reproductive Medicine
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• v.39 no.2
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• pp.87-93
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• 2012

Objective: Lin28 has been known to control the proliferation and pluripotency of embryonic stem cells. The purpose of this study was to determine the downstream effectors of Lin28 in mouse embryonic stem cells (mESCs) by RNA interference and microarray analysis. Methods: The control siRNA and Lin28 siRNA (Dharmacon) were transfected into mESCs. Total RNA was prepared from each type of transfected mESC and subjected to reverse transcription-polymerase chain reaction (RT-PCR) analysis to confirm the downregulation of Lin28. The RNAs were labeled and hybridized with an Affymetrix Gene-Chip Mouse Genome 430 2.0 array. The data analysis was accomplished by GenPlex 3.0 software. The expression levels of selected genes were confirmed by quantitative real-time RT-PCR. Results: According to the statistical analysis of the cDNA microarray, a total of 500 genes were altered in Lin28-downregulated mESCs (up-regulated, 384; down-regulated, 116). After differentially expressed gene filtering, 31 genes were selected as candidate genes regulated by Lin28 downregulation. Among them, neuropeptide Y5 receptor and oocyte-specific homeobox 5 genes were significantly upregulated in Lin28-downregulated mESCs. We also showed that the families of neuropeptide Y receptor (Npyr) and oocyte-specific homeobox (Obox) genes were upregulated by downregulation of Lin28. Conclusion: Based on the results of this study, we suggest that Lin28 controls the characteristics of mESCs through the regulation of effectors such as the Npyr and Obox families.

• Animal cells and systems
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• v.12 no.1
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• pp.15-21
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• 2008

Nitric oxide(NO) has been known to play important roles in numerous physiologic processes including neurotransmission, vasorelaxation, and cellular apoptosis. Using a mouse cDNA gene chip, we examined expression patterns and time course of NO-dependent genes in mouse macrophage RAW264.7 cells. Genes shown to be upregulated more than two fold or at least at two serial time points were further selected and validated by RT-PCR. Finally, 81 selected genes were classified by function as signaling, apoptosis, inflammation, transcription, translation, ionic homeostasis and metabolism. Among those, genes related with signaling, apoptosis and inflammation, such as guanylate cyclase 1, soluble, alpha3(Gucy1a3); protein kinase C, alpha($Pkc{\alpha}$); lymphocyte protein tyrosine kinase(Lck); BCL2/adenovirus E1B 19 kDa-interacting protein(Bnip3); apoptotic protease activating factor 1(Apaf1); X-linked inhibitor of apoptosis(Xiap); cyclin G1(Ccng1); chemokine(C-C motif) ligand 4(Ccl4); B cell translocation gene 2, anti-proliferative(Btg2); lysozyme 2(Lyz2); secreted phosphoprotein 1(Spp1); heme oxygenase(decycling) 1(Hmox1); CD14 antigen(Cd14); and granulin(Grn) may play important roles in NO-dependent responses in murine macrophages.