• ALGAE
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• v.31 no.2
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• pp.167-174
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• 2016

Biological response of cells to variable conditions should affect the expression level of certain genes. Quantification of these changes in target genes needs stable internal controls. Real-time quantitative polymerase chain reaction (PCR) has traditionally used reference or ‘housekeeping’ genes, that are considered to maintain equal expression in different conditions, to evaluate changes in target genes between samples and experimental conditions. Recent studies showed that some housekeeping genes may vary considerably in certain biological samples. This has not been evaluated in red algae. In order to identify the optimal internal controls for real-time PCR, we studied the expression of eleven commonly used housekeeping genes; elongation factor 1-alpha, glyceraldehyde-3-phosphate dehydrogenase, β-actin, polyubiquitin, 30S ribosomal gene, 60S ribosomal gene, beta-tubulin, alpha-tubulin, translation initiation factor, ubiquitin-conjugating enzyme, and isocitrate dehydrogenase in different life-history stages of Bostrychia moritziana. Our results suggest that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and 30S ribosomal gene, have the most stable gene expression levels between the different life history stages (male, female, carposporophyte, and tetrasporophyte), while the other genes are not satisfactory as internal controls. These results suggest that the combinations of GAPDH and 30S would be useful as internal controls to assess expression level changes in genes that may control different physiological processes in this organism or that may change in different life history stages. These results may also be useful in other red algal systems.

• Korean Journal of Veterinary Service
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• v.45 no.4
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• pp.305-316
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• 2022

Bordetella (B.) bronchiseptica, Mycoplasma (M.) felis, and Chlamydia (C.) felis are considered as main bacterial pathogens of feline upper respiratory tract disease (URTD). In this study, a new triplex quantitative real-time polymerase chain reaction (tqPCR) assay was developed for the rapid and differential detection of these bacteria in a single reaction. The assay specifically amplified three bacterial genes with the detection limit of below 10 copies/reaction. The assay showed high repeatability and reproducibility, with coefficients of intra-assay and inter-assay variation of less than 1%. Based on the diagnostic results of the assay using 94 clinical samples obtained from cats with URTD signs, prevalence of B. bronchiseptica, M. felis, or C. felis was 10.6%, 36.2%, or 6.4%, respectively, indicating that the diagnostic sensitivity was comparable to those of previously reported monoplex qPCR assays. The dual infection rates for B. bronchiseptica and M. felis or M. felis and C. felis was 2.1% or 3.2%, respectively. These results indicated that M. felis has been widely spread, and its co-infection with B. bronchiseptica or M. felis has been frequently occurred in Korean cat population. The developed tqPCR assay will serve as a promising tool for etiological and epidemiological studies of these three bacterial pathogens and the prevalence data obtained in this study will contribute to expanding knowledge about the epidemiology of feline URTD in Korea.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.11
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• pp.6949-6953
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• 2013

Background: Nowadays, the encapsulation of cytotoxic chemotherapeutic agents is attracting interest as a method for drug delivery. We hypothesized that the efficiency of helenalin might be maximized by encapsulation in ${\beta}$-cyclodextrin nanoparticles. Helenalin, with a hydrophobic structure obtained from flowers of Arnica chamissonis and Arnica Montana, has anti-cancer and anti-inflammatory activity but low water solubility and bioavailability. ${\beta}$-Cyclodextrin (${\beta}$-CD) is a cyclic oligosaccharide comprising seven D-glucopyranoside units, linked through 1,4-glycosidic bonds. Materials and Methods: To test our hypothesis, we prepared ${\beta}$-cyclodextrin-helenalin complexes to determine their inhibitory effects on telomerase gene expression by real-time polymerase chain reaction (q-PCR) and cytotoxic effects by colorimetric cell viability (MTT) assay. Results: MTT assay showed that not only ${\beta}$-cyclodextrin has no cytotoxic effect on its own but also it demonstrated that ${\beta}$-cyclodextrin-helenalin complexes inhibited the growth of the T47D breast cancer cell line in a time and dose-dependent manner. Our q-PCR results showed that the expression of telomerase gene was effectively reduced as the concentration of ${\beta}$-cyclodextrin-helenalin complexes increased. Conclusions: ${\beta}$-Cyclodextrin-helenalin complexes exerted cytotoxic effects on T47D cells through down-regulation of telomerase expression and by enhancing Helenalin uptake by cells. Therefore, ${\beta}$-cyclodextrin could be superior carrier for this kind of hydrophobic agent.

• Journal of Microbiology and Biotechnology
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• v.30 no.10
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• pp.1495-1499
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• 2020

The study of climate and respiratory viral infections using big data may enable the recognition and interpretation of relationships between disease occurrence and climatic variables. In this study, real-time reverse transcription quantitative PCR (qPCR) methods were used to identify Human respiratory coronaviruses (HCoV). infections in patients below 10 years of age with respiratory infections who visited Dankook University Hospital in Cheonan, South Korea, from January 1, 2012, to December 31, 2018. Out of the 9010 patients who underwent respiratory virus real-time reverse transcription qPCR test, 364 tested positive for HCoV infections. Among these 364 patients, 72.8% (n = 265) were below 10 years of age. Data regarding the frequency of infections was used to uncover the seasonal pattern of the two viral strains, which was then compared with local meteorological data for the same time period. HCoV-229E and HCoV-OC43 showed high infection rates in patients below 10 years of age. There was a negative relationship between HCoV-229E and HCoV-OC43 infections with air temperature and wind-chill temperatures. Both HCoV-229E and HCoV-OC43 rates of infection were positively related to atmospheric pressure, while HCoV-229E was also positively associated with particulate matter concentrations. Our results suggest that climatic variables affect the rate in which children below 10 years of age are infected with HCoV. These findings may help to predict when prevention strategies may be most effective.

• Korean Journal of Poultry Science
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• v.44 no.3
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• pp.199-209
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• 2017

Mitogen-activated protein kinase (MAPK) signaling pathways play a key role in innate immunity, inflammation, cell proliferation, cell differentiation, and cell death. The main objective of this study was to investigate the expression level of candidate MAPK pathway genes in the intestinal mucosal layer of two genetically disparate chicken lines (Marek's disease-resistant line 6.3 and Marek's disease-susceptible line 7.2) induced with necrotic enteritis (NE). Using high-throughput RNA sequencing, we investigated 178 MAPK signaling pathway related genes that were significantly and differentially expressed between the intestinal mucosal layers of the NE-afflicted and control chickens. In total, 15 MAPK pathway genes were further measured by quantitative real-time PCR(qRT-PCR) and the results were consistent with the RNA-sequencing data. All 178 identified genes were annotated through Gene Ontology and mapped onto the KEGG chicken MAPK signaling pathway. Several key genes of the MAPK pathway, ERK1/2, JNK1-3, p38 MAPK, MAP2K1-4, $NF-{\kappa}B1/2$, c-Fos, AP-1, Jun-D, and Jun, were differentially expressed in the two chicken lines. Therefore, we believe that RNA sequencing and qRT-PCR analysis provide resourceful information for future studies on MAPK signaling of genetically disparate chicken lines in response to pathogens.

• Asian Pacific Journal of Cancer Prevention
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• v.17 no.12
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• pp.5229-5235
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• 2016

Prostate cancer (PCa) remains one of the most widespread and perplexing of all human malignancies. Assessment of gene expression is thought to have an important impact on cancer diagnosis, prognosis and therapeutic decisions. In this context, we explored combined expression of PCa related target genes AMACR and PCA3 in 126 formalin fixed paraffin embedded prostate tissues (FFPE) from Moroccan patients, using quantitative real time reverse transcription-PCR (RT-qPCR). This quantification required data normalization accomplished using stably expressed reference genes (RGs). A panel of twelve RG was assessed, data being analyzed using GenEx V6 based on geNorm, NormFinder and statistical methods. Accordingly, the hnRNP A1 gene was identified and selected as the most stably expressed RG for reliable and accurate gene expression quantification in prostate tissues. The ratios of both PCA3 and AMACR gene expression relative to that of the hnRNP A1 gene were calculated and the performance of each target gene for PCa diagnosis was evaluated using receiver-operating characteristics. PCA3 and AMACR mRNA quantification based on RT-qPCR may prove useful in PCa diagnosis. Of particular interesting, combining PCA3 and AMACR quantification improved PCa prediction by increasing sensitivity with retention of good specificity.

• Genes and Genomics
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• v.40 no.11
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• pp.1157-1167
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• 2018

Sulfolobus species can grow on a variety of organic compounds as carbon and energy sources. These species degrade glucose to pyruvate by the modified branched Entner-Doudoroff pathway. We attempted to determine the differentially expressed genes (DEGs) under sugar-limited and sugar-rich conditions. RNA sequencing (RNA-seq) was used to quantify the expression of the genes and identify those DEGs between the S. acidocaldarius cells grown under sugar-rich (YT with glucose) and sugar-limited (YT only) conditions. The functions and pathways of the DEGs were examined using gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. Quantitative real-time PCR (qRT-PCR) was performed to validate the DEGs. Transcriptome analysis of the DSM 639 strain grown on sugar-limited and sugar-rich media revealed that 853 genes were differentially expressed, among which 481 were upregulated and 372 were downregulated under the glucose-supplemented condition. In particular, 70 genes showed significant changes in expression levels of ${\geq}$ twofold. GO and KEGG enrichment analyses revealed that the genes encoding components of central carbon metabolism, the respiratory chain, and protein and amino acid biosynthetic machinery were upregulated under the glucose condition. RNA-seq and qRT-PCR analyses indicated that the sulfur assimilation genes (Saci_2197-2204) including phosphoadenosine phosphosulfate reductase and sulfite reductase were significantly upregulated in the presence of glucose. The present study revealed metabolic networks in S. acidocaldarius that are induced in a glucose-dependent manner, improving our understanding of biomass production under sugar-rich conditions.

• Entomological Research
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• v.48 no.5
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• pp.390-399
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• 2018

In order to precisely assess gene expression levels, the suitable internal reference genes must be served to quantify real-time reverse transcription polymerase chain reaction (RT-qPCR) data. For armyworm, Mythimna separata, which reference genes are suitable for assessing the level of transcriptional expression of target genes have yet to be explored. In this study, eight common reference genes, including ${\beta}$-actin (${\beta}$-ACT), 18 s ribosomal (18S), 28S ribosomal (28S), glyceraldehyde-3-phosphate (GAPDH), elongation fator-alpha ($EF1{\alpha}$), TATA box binding protein (TBP), ribosomal protein L7 (RPL7), and alpha-tubulin (${\alpha}$-TUB) that in different developmental stages, tissues and insecticide treatments of M. separata were evaluated. To further explore whether these genes were suitable to serve as endogenous controls, three software-based approaches (geNorm, BestKeeper, and NormFinder), the delta Ct method, and one web-based comprehensive tool (RefFinder) were employed to analyze and rank the tested genes. The optimal number of reference genes was determined using the geNorm program, and the suitability of particular reference genes was empirically validated according to normalized HSP70, and MsepCYP321A10 gene expression data. We found that the most suitable reference genes for the different experimental conditions. For developmental stages, 28S/RPL7 were the optimal reference genes, both $RPL7/EF1{\alpha}$ were suitable for experiments of different tissues, whereas for insecticide treatments, $28S/{\alpha}-TUB$ were suitable for normalizations of expression data. In addition, $28S/{\alpha}-TUB$ were the suitable reference genes because they have the most stable expression among different developmental stages, tissues and insecticide treatments. Our work is the first report on reference gene selection in M. separata, and might serve as a precedent for future gene expression studies.

• Journal of Apiculture
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• v.34 no.1
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• pp.39-46
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• 2019

BQCV multi-point PCR was developed as a rapid multiplex detection method for BQCV, one of the viral pathogens of honeybees. It could detect BQCV specific genes qualitative as well as quantitative detection based on ultra-rapid PCR. Three primer pairs (RNA dependent RNA polymerase, capsid protein, 3C like protease) were specifically designed for accurate the detection and were optimized for minimizing the detection time and increasing the sensitivity. Our advanced diagnostic system have the accuracy by lowering the concern about the variation in the BQCV detection site. In addition, it should be an opportunity to identify mutations that are mixed with other viruses.

• BMB Reports
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• v.42 no.9
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• pp.593-598
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• 2009

Cell cycle progression is regulated by both transcriptional and post-transcriptional mechanisms. MicroRNAs (miRNAs) emerge as a new class of small non-coding RNA regulators of cell cycle as recent evidence suggests. It is hypothesized that expression of specific miRNAs oscillates orderly along with cell cycle progression. However, the oscillated expression patterns of many candidate miRNAs have yet to be determined. Here, we describe miRNA expression profiling in double-thymidine synchronized HeLa cells as cell cycle progresses. Twenty-five differentially expressed miRNAs were classified into five groups based on their cell cycle-dependent expression patterns. The cyclic expression of six miRNAs (miR-221, let-7a, miR-21, miR-34a, miR-24, miR-376b) was validated by real-time quantitative RT-PCR (qRT-PCR). These results suggest that specific miRNAs, along with other key factors are required for maintaining and regulating proper cell cycle progression. The study deepens our understanding on cell cycle regulation.