• Communications for Statistical Applications and Methods
• /
• v.16 no.6
• /
• pp.1037-1046
• /
• 2009

Copy number variations(CNVs) are known as one of the most important factors in susceptibility to genetic disorders because they affect expression levels of genes. In previous studies, pyrosequencing, mini-sequencing real-time polymerase chain reaction(PCR), invader assays and other techniques have been used to detect CNVs. However, the higher the copy number in a genome, the more difficult it is to resolve the copies, so a more accurate method for measuring CNVs and assigning genotype is needed. PCR followed by a quantitative oligonucleotide ligation assay(qOLA) was developed for quantifying CNVs. The aim of this study was to compare the two methods for detecting and quantifying the CNVs of duplicated gene: the published pyrosequencing assay(pyro_CNV) and the newly developed qOLA_CNV. The accuracy and precision of the assay were evaluated for porcine KIT, which was selected as a model locus. Overall, the root mean squares(RMSs) of bias and standard deviation of qOLA_CNV were 2.09 and 0.45, respectively. These values are less than half of those of pyro CNV.

• International Journal of Industrial Entomology and Biomaterials
• /
• v.23 no.1
• /
• pp.137-141
• /
• 2011

A previous data was provided information for tissuespecific expression genes by means of whole-genome oligonucleotide microarray in the silkworm. We analyzed the tissue-specific expression patterns in the hemocyte tissue on 5 days of 5th instar larvae during the development of $B.$ $mori$. Total 5 candidates pick out from the $Bombyx$ $mori$ Microarray Database (BmMDB; http://silkworm.swu.edu.cn/microarray). To verify the hemocyte-specific expression, we analyzed by semi-quantitative and real-time quantitative RT-PCR using the highly expressed endogenous $Actin$ RNA as an intrinsic reference. In this study, we confirmed that one gene-sw17255- out of 5 candidates expressed in the hemocyte tissue, which was consistent with the previous data. Circulating hemocytes in the body fluid of the $B.$ $mori$ are most powerful target organ for producing biomaterials. We need further studies to find hemocyte-specific promoter region from sw17255 gene. Finally, this result can be applied in creating transgenic silkworms as a biomedical insect.

• The Journal of Korean Medicine
• /
• v.21 no.1
• /
• pp.53-61
• /
• 2000

Objectives: This study was camed out to examine the effect of five fractions of aqueous extract from Artemisia capillaris THUNB(ACT), on TGF, 1-induced apoptosis, cell viability, cell cycle progression and mRNA expression of apoptosis-related genes in human hepatocyte cell line HepG2. Methods: This study employed Tryphan blue exclusion assay, DNA fragmentation assay, Cpp32 protease activity assay and Quantitative RT-PCR analysis. Results: In the Tryphan blue exclusion assay, the butanol fraction of ACT with $TGF{\beta}$, l showed magnificent (Nice word, ut is it appropriate in a medical abstract\ulcorner) viability and the H2O fraction of ACT with $TGF{\beta}$, l also showed higher viability than only $TGF{\beta}$, l-treated group. DNA fragmentation assay showed that the butanol fraction and the H2O fraction carried inhibitory effects on apoptosis induction, with the butanol fraction displaying greater effects. The Cpp32 protease activity assay showed that the butanol fraction decreased Cpp32 protease activity. The H2O fraction of ACT had no significant effect on the Cpp32 protease activity. Quantitative RT-PCR showed that the butanol fraction suppressed Bax, p 15/INK4B, p21/Waf1, PAI-1 and increased Bcl-2 gene. Conclusions: The data shows that butanol fraction of ACT increases the hepatocyte viability and has the hepatocellular protective effect by the suppression of $TGF{\beta}$, l induced-apoptosis through gene regulation.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
• /
• v.41 no.1
• /
• pp.11-18
• /
• 2015

Objectives: The goal of this study was to determine the correlation of clinicopathological factors and the up-regulation of vascular endothelial growth factor (VEGF) expression in oral squamous cell carcinoma. Materials and Methods: Immunohistochemical staining of VEGF and quantitative real-time polymerase chain reaction (RT-PCR) of VEGF mRNA were performed in 20 specimens from 20 patients with oral squamous cell carcinoma and another 20 specimens from 20 patients with carcinoma in situ as a controlled group. Results: The results were as follows: 1) In immunohistochemical study of poorly differentiated and invasive oral squamous cell carcinoma, high-level staining of VEGF was observed. Significant correlation was observed between immunohistochemical VEGF expression and histologic differentiation, tumor size of specimens (Pearson correlation analysis, significance r>0.6, P<0.05). 2) In VEGF quantitative RT-PCR analysis, progressive cancer showed more VEGF expression than carcinoma in situ. Paired-samples analysis determined the difference of VEGF mRNA expression level between cancer tissue and carcinoma in situ tissue, between T1 and T2-4 (Student's t-test, P<0.05). Conclusion: These findings suggest that up-regulation of VEGF may play a role in the angiogenesis and progression of oral squamous cell carcinoma.

• Journal of Pharmacopuncture
• /
• v.3 no.1
• /
• pp.1-19
• /
• 2000

To study anti-cancer effect and molecular biological mechanism of bee venom for aqua-acupuncture, the effects of bee venom on cell viability and apoptosis were analyzed using MTT assay, tryphan blue assay, $[^3H]$thymidine release assay, flow cytometric analysis, and activity of caspase-3 protease activity assay. To explore whether anti-cancer effects of bee venom are associated with the transcriptional control of gene expression, quantitative RT-PCR analysis of apoptosis-related genes was performed. The obtained results are summarized as follows: 1. The MTT assay demonstrated that cell viability was decreased by bee venom in a dose-dependant manner. 2. Significant induction of apoptosis was identified using tryphan blue assay, $[^3H]$thymidine release assay, and flow cytomet1 ric analysis of sub $G_1$ fraction. 3. In analysis of caspase-3 protease activity, the activity had increased significantly, in a dose-dependant manner. 4. Quantitative RT-PCR analysis of the apoptosis-related genes showed that Bcl-2 and Bcl-$X_L$ were down-regulated whereas Bax was up-regulated by bee venom treatment.

• Tuberculosis and Respiratory Diseases
• /
• v.72 no.3
• /
• pp.293-301
• /
• 2012

Background: Ventilator-associated pneumonia (VAP) requires prompt and appropriate treatment. Since methicillin-resistant Staphylococcus aureus (MRSA) is a frequent pathogen in VAP, rapid identification of it, is pivotal. Our aim was to evaluate the utility of quantitative polymerase chain reaction (qPCR) as a useful method for etiologic diagnoses of MRSA pneumonia. Methods: We performed qPCR for mecA, S. aureus-specific femA-SA, and S. epidermidis-specific femA-SE genes from bronchoalveolar lavage or bronchial washing samples obtained from clinically-suspected VAP. Molecular identification of MRSA was based on the presence of the mecA and femA-SA gene, with the absence of the femA-SE gene. To compensate for the experimental and clinical conditions, we spiked an internal control in the course of DNA extraction. We estimated number of colony-forming units per mL (CFU/mL) of MRSA samples through a standard curve of a serially-diluted reference MRSA strain. We compared the threshold cycle (Ct) value with the microbiologic results of MRSA. Results: We obtained the mecA gene standard curve, which showed the detection limit of the mecA gene to be 100 fg, which corresponds to a copy number of 30. We chose cut-off Ct values of 27.94 (equivalent to $1{\times}10^4$ CFU/mL) and 21.78 (equivalent to $1{\times}10^5$ CFU/mL). The sensitivity and specificity of our assay were 88.9% and 88.9% respectively, when compared with quantitative cultures. Conclusion: Our results were valuable for diagnosing and identifying pathogens involved in VAP. We believe our modified qPCR is an appropriate tool for the rapid diagnosis of clinical pathogens regarding patients in the intensive care unit.

• Journal of fish pathology
• /
• v.31 no.2
• /
• pp.93-99
• /
• 2018

Edwardsiella tarda predominantly causes edwardsiellosis in fish at high temperature, but is rarely isolated from water when water temperature is low. However, E. tarda is viable but nonculturable (VBNC) in low water temperature, but it can be revived when water temperature rises and cause disease to fish. Therefore, in order to prevent disease, it is very important to identify pathogens that are in the VBNC state in environmental water. In this study, E. tarda cells in the VBNC state were detected by the ethidium monoazide (EMA)-PCR method using the low-temperature oligotrophic sea water microcosm obtained by inoculation of E. tarda at a concentration of $10^8CFU/ml$. In order to distinguish between live and dead bacteria in E. tarda, each sample was treated with EMA at different concentrations, photoactivated with a 500 W halogen lamp, and PCR was performed with E. tarda specific primer. At the concentration of $10^7CFU/ml$ bacterium, DNA amplification was observed only in the live cells when treated with $60{\mu}g/ml$ of EMA, and smaller amounts of live cells could be distinguished from dead cells by adjusting the EMA concentration. In addition, the VBNC cells of E. tarda in the oligotrophic low temperature seawater microcosm were estimated to be in the range of $10^4{\sim}10^5CFU/ml$ by EMA-PCR. Therefore, it is possible to detect VBNC cells that will act as potential pathogens in environmental water using EMA-PCR method, and quantitative confirmation using concentration change is also possible.

• Journal of Microbiology and Biotechnology
• /
• v.31 no.12
• /
• pp.1709-1715
• /
• 2021

Outbreaks of food poisoning due to the consumption of norovirus-contaminated shellfish continue to occur. Male-specific (F+) coliphage has been suggested as an indicator of viral species due to the association with animal and human wastes. Here, we compared two methods, the double agar overlay and the quantitative real-time PCR (RT-PCR)-based method, for evaluating the applicability of F+ coliphage-based detection technique in microbial contamination tracking of shellfish samples. The RT-PCR-based method showed 1.6-39 times higher coliphage PFU values from spiked shellfish samples, in relation to the double agar overlay method. These differences indicated that the RT-PCR-based technique can detect both intact viruses and non-particle-protected viral DNA/RNA, suggesting that the RT-PCR based method could be a more efficient tool for tracking microbial contamination in shellfish. However, the virome information on F+ coliphage-contaminated oyster samples revealed that the high specificity of the RT-PCR- based method has a limitation in microbial contamination tracking due to the genomic diversity of F+ coliphages. Further research on the development of appropriate primer sets for microbial contamination tracking is therefore necessary. This study provides preliminary insight that should be examined in the search for suitable microbial contamination tracking methods to control the sanitation of shellfish and related seawater.

• Journal of Microbiology and Biotechnology
• /
• v.12 no.5
• /
• pp.801-806
• /
• 2002

A multiplex competitive polymerase chain reaction (PCR) method was developed for the rapid identification and quantification of Leuconostoc mesnteroides and Lactobacillus plantarum populations which are the key microorganisms in kimchi fermentation. The strain-specific primers were designed to selectively amplify the target genes encoding 165 rRNA of L. plantarum and dextransucrase of L. mesenteroides. There was a linear relationship between the band intensity of PCR products and the number of colony forming units of each model organism. The PCR quantification method was compared with a traditional plate-counting method f3r the enumeration of the two lactic acid bacteria in a mixed suspension culture and also applied to a real food system, namely, watery kimchi. The population dynamics of the two model organisms in the mixed culture were reliably predictable by the competitive PCR analysis.

• Reproductive and Developmental Biology
• /
• v.33 no.4
• /
• pp.249-255
• /
• 2009

The miniature pig is considered to be a better organ donor breed for xenotransplantation than other pig breeds because the size of the organs of the miniature pig is similar to that of humans. In this study, we aimed at identifying differentially expressed genes in the miniature pig ovary during pregnancy. For this, we used the miniature pig ovary model, annealing control primer-based reverse transcription polymerase chain reaction (PCR), quantitative real-time PCR (qRT-PCR), and northern blotting analysis. We identified 13 genes showing differential expression on the based of pregnancy status and validated 8 genes using qRT-PCR. We also sequenced the full-length cDNA of ephrin receptor A4 (EphA4), which had a significant difference in expression level, and validated it by northern blotting. These genes may provide a better understanding of the cellular and molecular mechanisms during pregnancy in miniature pig ovary.