• Journal of Marine Life Science
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• v.1 no.2
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• pp.79-87
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• 2016

Heavy metals such as cadmium (Cd) are highly toxic to aquatic organisms and human, even at trace concentration. Herein we investigated the effect of Cd on the gene expression of ATP-binding cassette (ABC) transporters and glutathione S-transferase (GST) in marine ciliate Euplotes crassus. Seven ABC transporters and one GST genes were partially cloned and sequences, and thereafter, transcriptional modulation of these genes after exposure to Cd for 8 h was investigated using quantitative real time RT- PCR (qRT-PCR). As results, sequence analysis and phylogenetic study revealed that E. crassus ABCs are likely typical ABC transports, in particular, B/C family, and GST gene may be similar to GST theta isoform. A significant increase in the expression of ABCs, except for ABCB21 was observed in a concentration dependent manner after exposure to Cd (0.1 and 0.5 mg/l) for 8 h. The GST mRNA level was the highest at 0.5 mg/l Cd and then reduced until control level. These findings suggest that ABCs and GST may be involved in a protective mechanism against Cd-mediated toxicity in E. crassus.

• Reproductive and Developmental Biology
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• v.35 no.1
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• pp.1-8
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• 2011

Techniques to evaluate gene expression profiling, such as sufficiently sensitive cDNA microarrays or real-time quantitative PCR, are efficient methods for monitoring human pluripotent stem cell (hESC/iPSC) cultures. However, most of these high-throughput tests have a limited use due to high cost, extended turn-around time, and the involvement of highly specialized technical expertise. Hence, there is an urgency of rapid, cost-effective, robust, yet sensitive method development for routine screening of hESCs/hiPSCs. A critical requirement in hESC/hiPSC cultures is to maintain a uniform undifferentiated state and to determine their differentiation capacity by showing the expression of gene markers representing all three germ layers, including ectoderm, mesoderm, and endoderm. To quantify the modulation of gene expression in hESCs/hiPSC during their propagation, expansion, and differentiation via embryoid body (EB) formation, we developed a simple, rapid, inexpensive, and definitive multimarker, semiquantitative multiplex RT-PCR platform technology. Among the 9 gene primers tested, 5 were pluripotent markers comprising set 1, and 3 lineage-specific markers were combined as set 2, respectively. We found that these 2 sets were not only effective in determining the relative differentiation in hESCs/hiPSCs, but were easily reproducible. In this study, we used the hES/hiPS cell lines to standardize the technique. This multiplex RT-PCR assay is flexible and, by selecting appropriate reporter genes, can be designed for characterization of different hESC/hiPSC lines during routine maintenance and directed differentiation.

• The Plant Pathology Journal
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• v.39 no.4
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• pp.309-318
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• 2023

Huanglongbing (HLB) is one of the most destructive diseases in citrus, which imperils the sustainability of citriculture worldwide. The presumed causal agent of HLB, 'Candidatus Liberibacter asiaticus' (CLas) is a non-culturable phloem-limited α-proteobacterium transmitted by Asian citrus psyllids (ACP, Diaphorina citri Kuwayama). A widely adopted method for HLB diagnosis is based on quantitative real-time polymerase chain reaction (qPCR). Although HLB diagnostic qPCR provides high sensitivity and good reproducibility, it is limited by time-consuming DNA preparation from plant tissue or ACP and the requirement of proper lab instruments including a thermal cycler to conduct qPCR. In an attempt to develop a quick assay that can be deployed in the field for CLas detection, we developed a real-time loop-mediated isothermal amplification (rt-LAMP) assay by targeting the CLas five copy nrdB gene. The rt-LAMP assay using various plant sample types and psyllids successfully detected the nrdB target as low as ~2.6 Log10 copies. Although the rt-LAMP assay was less sensitive than laboratory-based qPCR (detection limit ~10 copies), the data obtained with citrus leaf and bark and ACP showed that the rt-LAMP assay has >96% CLas detection rate, compared to that of laboratory-based qPCR. However, the CLas detection rate in fibrous roots was significantly decreased compared to qPCR due to low CLas titer in some root DNA sample. We also demonstrated that the rt-LAMP assay can be used with a crude leaf DNA extract which is fully deployable in the field for quick and reliable HLB screening.

• Journal of Food Hygiene and Safety
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• v.33 no.1
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• pp.71-76
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• 2018

Norovirus is a leading cause of sporadic pathogenic non-bacterial gastroenteritis worldwide. For the detection of norovirus, reverse transcription real-time PCR (RT qPCR) has quickly become a major tool due to its sensitivity and specificity. However, accurate viral RNA extraction methods are essential for RT qPCR analysis. TRIzol reagents are used to extract RNA from biological materials and are therefore widely used for norovirus RNA extraction. In this study, the yield of viral RNA extraction using TRIzol from genogroup II (GII) among the human norovirus genogroup I (GI) and GII, and murine norovirus (GV) depended on the pH of the virus sample solution. The yield of RNA extraction was higher at the alkaline pH than in the acidic region compared with the Ct (threshold cycle) value of the real-time PCR. From the results of this study, it was found that the pH condition is very important for the quantitative analysis of norovirus by extracting GII RNA using TRIzol.

• Journal of the Society of Cosmetic Scientists of Korea
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• v.39 no.4
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• pp.281-288
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• 2013

Hyaluronic acid (HA) is one of the major extracellular matrix components in skin. The HA content is reported to decline with age, which may contribute to decrease in skin moisture, wrinkle formation and the decrease in elasticity of the skin. Among the family of HA synthase genes (HAS-1, 2, 3) identified so far, HAS-2 plays crucial roles in the regulation of HA synthesis in human skin fibroblasts. In this study, we elucidated the effects of ferulic acid isolated from Cnidium officinale on HA production. Semi-quantitative RT-PCR and quantitative real-time PCR showed that ferulic acid increased mRNA level of HAS-2 gene and ELISA assay also revealed that ferulic acid increased HA production in human skin fibroblasts. Our study suggests that ferulic acid might prevent age-dependent skin deteriorations such as wrinkles, dryness and elasticity decrease, all of which could be ascribed to the reduction of the HA content in human skin.

• Asian-Australasian Journal of Animal Sciences
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• v.20 no.2
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• pp.184-193
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• 2007

In mammalian ovary, steroidogenic acute regulatory (StAR) protein mediates the true rate-limiting step of transport of cholesterol from outer to inner mitochondrial membrane. Appropriate expression of StAR gene represents an indispensable component of steroidogenesis and its regulation has been found to be species specific. However, limited information is available regarding StAR gene expression during estrous cycle in buffalo ovary. In the present study, expression, localization and hormonal regulation of StAR mRNA were analyzed by semi-quantitative RT-PCR in buffalo ovary and partial cDNA was cloned. Total RNA was isolated from whole follicles of different sizes, granulosa cells from different size follicles and postovulatory structures like corpus luteum and Corpus albicans. Semi-quantitative RT-PCR analyses showed StAR mRNA expression in the postovulatory structure, corpus luteum. No StAR mRNA was detected in total RNA isolated from whole follicles of different size including the preovulatory follicle (>9 mm in diameter). However, granulosa cells isolated from preovulatory follicles showed the moderate expression of StAR mRNA. To assess the hormonal regulation of StAR mRNA, primary culture of buffalo granulosa cells were treated with FSH (100 ng/ml) alone or along with IGF-I (100 ng/ml) for 12 to 18 h. The abundance of StAR mRNA increased in cells treated with FSH alone or FSH with IGF-I. However, effect of FSH with IGF-I on mRNA expression was found highly significant (p<0.01). In conclusion, differential expression of StAR messages was observed during estrous cycle in buffalo ovary. Also, there was a synergistic action of IGF-I on FSH stimulation of StAR gene.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.4
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• pp.1675-1679
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• 2014

Objectives: MicroRNAs (miRNAs) are important regulators of many physiological and pathological processes, including tumorigenesis and metastasis. In this study, we sought to determine the underlying molecular mechanisms of metastatic cervical carcinoma by performing miRNA profiling. Methods: Tissue samples were collected from ten cervical squamous cancer patients who underwent hysterectomy and pelvic lymph node (PLN) dissection in our hospital, including four PLN-positive (metastatic) cases and six PLN-negative (non-metastatic) cases. A miRNA microarray platform with 1223 probes was used to determine the miRNA expression profiles of these two tissue types and case groups. MiRNAs having at least 4-fold differential expression between PLN-positive and PLN-negative cervical cancer tissues were bioinformatically analyzed for target gene prediction. MiRNAs with tumor-associated target genes were validated by quantitative reverse transcription-polymerase chain reaction (RT-PCR). Results: Thirty-nine miRNAs were differentially expressed (>4-fold) between the PLN-positive and PLN-negative groups, of which, 22 were up-regulated and 17 were down-regulated. Sixty-nine percent of the miRNAs (27/39) had tumor-associated target genes, and the expression levels of six of those (miR-126, miR-96, miR-144, miR-657, miR-490-5p, and miR-323-3p) were confirmed by quantitative (q)RT-PCR. Conclusions: Six MiRNAs with predicted tumor-associated target genes encoding proteins that are known to be involved in cell adhesion, cytoskeletal remodeling, cell proliferation, cell migration, and apoptosis were identified. These findings suggest that a panel of miRNAs may regulate multiple and various steps of the metastasis cascade by targeting metastasis-associated genes. Since these six miRNAs are predicted to target tumor-associated genes, it is likely that they contribute to the metastatic potential of cervical cancer and may aid in prognosis or molecular therapy.

• Journal of Genetic Medicine
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• v.19 no.2
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• pp.63-75
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• 2022

Purpose: Osteoporosis is a common calcium and metabolic skeletal disease which is characterized by decreased bone mass, microarchitectural deterioration of bone tissue and impaired bone strength, thereby leading to enhanced risk of bone fragility. In this study, we aimed to identify novel genes for susceptibility to osteoporosis and/or bone density. Materials and Methods: To identify differentially expressed genes (DEGs) between control and osteoporosis-induced cells, annealing control primer-based differential display reverse-transcription polymerase chain reaction (RT-PCR) was carried out in pre-osteoblast MC3T3-E1 cells. Expression levels of the identified DEGs were evaluated by quantitative RT-PCR. Association studies for the quantitative bone density analysis and osteoporosis case-control analysis of single nucleotide polymorphism (SNPs) were performed in Korean women (3,570 subjects) from the Korean Association REsource (KARE) study cohort. Results: Comparison analysis of expression levels of the identified DEGs by quantitative RT-PCR found seven genes, Anxa6, Col5a1, Col6a2, Eno1, Myof, Nfib, and Scara5, that showed significantly different expression between the dexamethason-treated and untreated MC3T3-E1 cells and between the ovariectomized osteoporosis-induced mice and sham mice. Association studies revealed that there was a significant association between the SNPs in the five genes, ANXA6, COL5A1, ENO1, MYOF, and SCARA5, and bone density and/or osteoporosis. Conclusion: Using a whole-genome comparative expression analysis, gene expression evaluation analysis, and association analysis, we found five genes that were significantly associated with bone density and/or osteoporosis. Notably, the association P-values of the SNPs in the ANXA6 and COL5A1 genes were below the Bonferroni-corrected significance level.

• Applied Chemistry for Engineering
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• v.33 no.3
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• pp.235-241
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• 2022

According to the World Water Development Report 2015 released by the United Nations, drinking water is expected to decrease by 40% by 2030. This does not mean that the amount of water decreases, but rather that the water source is contaminated due to environmental pollution. Because microbes are deeply related to water quality, the analysis of microbe is very important for water quality management. While the most common method currently used for microbial analysis is microscopic examination of the shape and feature after cell culture, as the gene analysis technology advances, quantitative polymerase chain reaction (qPCR) can be applied to the microscopic microbiological analysis, and the application method has been studied. Among them, a reverse transcription (RT) step enables the analysis of RNA by RT-PCR. Integrated cell culture (ICC)-qPCR shortens the test time by using it with microbial culture analysis, and viability qPCR can reduce the false positive errors of samples collected from natural water source. Multiplex qPCR for improved throughput, and microfluidic qPCR for analysis with limited amount of sample has been developed In this paper, we introduce the case, principle and development direction of the qPCR method applied to the analysis of microorganisms.

• Journal of the korean academy of Pediatric Dentistry
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• v.45 no.1
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• pp.32-40
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• 2018

The aim of this study was to determine the efficacy of a 3 tone plaque disclosing gel in assessing the risk of caries related to the population of Streptococcus mutans, Streptococcus sobrinus, and Lactobacillus spp. quantified using a quantitative real-time polymerase chain reaction (qRT-PCR). 15 healthy children of ages 9 - 12 years were randomly examined. The 3 tone plaque disclosing gel was applied on teeth surfaces, which changed the color to pink or red, blue or purple and light blue. Plaque was divided into 3 groups based on staining. Genomic DNA from each sample was subjected to a qRT-PCR assay for quantitative detection of target bacteria. The Kruskal-Wallis test was conducted for correlation between the color of plaque and the number of bacterial species. The levels of S. mutans, S. sobrinus, and Lactobacillus spp. were significantly different in the plaque samples of the 3 groups (p < 0.05). The proportion of S. sobrinus to S. mutans showed correlation to the color of plaque. The different color-dyed plaque was related to the number of acidogenic bacteria. The 3 tone plaque disclosing gel could be used as one of the indicators to assess the clinical risk of caries associated with the population of S. mutans, S. sobrinus, and Lactobacillus spp.