• Journal of Microbiology and Biotechnology
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• v.27 no.1
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• pp.112-121
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• 2017

Edwardsiella tarda is a gram-negative pathogenic bacterium in aquaculture that can cause hemorrhagic septicemia in fish. Many secreted proteins have already been identified as virulent factors of E. tarda. Moreover, since virulent phenotypes are based on the expression regulation of virulent genes, understanding the expression profile of virulent genes is important. A quantitative RT-PCR is one of the preferred methods for determining different gene expressions. However, this requires the selection of a stable reference gene in E. tarda, which has not yet been systematically studied. Accordingly, this study evaluated nine candidate reference genes (recA, uup, rpoB, rho, topA, gyrA, groEL, rpoD, and 16S rRNA) using the Excel-based programs BestKeeper, GeNorm, and NormFinder under different culture conditions. The results showed that 16S rRNA was more stable than the other genes at different culture growth phases. However, at the same culture time, topA was identified as the reference gene under the conditions of different strains, different culture media, and infection, whereas gyrA was identified under the condition of different temperatures. Thus, in experiments, the expression of gapA and fbaA in E. tarda was analyzed by RT-qPCR using 16S rRNA, recA, and uup as the reference genes. The results showed that 16S rRNA was the most suitable reference gene in this analysis, and that using unsuitable reference genes resulted in inaccurate results.

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

• Parasites, Hosts and Diseases
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• v.51 no.4
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• pp.413-419
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• 2013

The mainstay therapy against leishmaniasis is still pentavalent antimonial drugs; however, the rate of antimony resistance is increasing in endemic regions such as Iran. Understanding the molecular basis of resistance to antimonials could be helpful to improve treatment strategies. This study aimed to recognize genes involved in antimony resistance of Leishmania tropica field isolates. Sensitive and resistant L. tropica parasites were isolated from anthroponotic cutaneous leishmaniasis patients and drug susceptibility of parasites to meglumine antimoniate (Glucantime$^{(R)}$) was confirmed using in vitro assay. Then, complementary DNA-amplified fragment length polymorphism (cDNA-AFLP) and real-time reverse transcriptase-PCR (RT-PCR) approaches were utilized on mRNAs from resistant and sensitive L. tropica isolates. We identified 2 known genes, ubiquitin implicated in protein degradation and amino acid permease (AAP3) involved in arginine uptake. Also, we identified 1 gene encoding hypothetical protein. Real-time RT-PCR revealed a significant upregulation of ubiquitin (2.54-fold), and AAP3 (2.86-fold) (P<0.05) in resistant isolates compared to sensitive ones. Our results suggest that overexpression of ubiquitin and AAP3 could potentially implicated in natural antimony resistance.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.33 no.6
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• pp.636-642
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• 2007

Background: CpG DNA plays an important role in immune cell function. This study examined whether the temporal control of toll-like receptor (TLR)9 by CpG DNA can regulate the expression of matrix metalloproteinase-9(MMP-9). Methods and materials: Macrophages were cultured in the presence of 10% FBS. For the various MMP genes analysis, RT-PCR and real-time PCR were performed. In addition, zymography assay performed for the MMP activity. The phosphorylation assay did for the ERK1/2 and NF${\kappa}B$ activation, and luciferase promoter assay was for the NF${\kappa}B$ activity. Results: CpG DNA induced the mRNA expression of MMP-2, MMP-9, and MMP-13, but not of MMP-7, MMP-8, and MMP-12, in a time-dependent manner. Especially, the mRNA expression of MMP-9 was strongly induced by CpG DNA using real-time RT-PCR. The TLR9 inhibitor, chloroquine, suppressed CpG DNA-induced MMP-9 expression and its activity. Moreover, CpG DNA induced the phosphorylation of ERK and the inhibition of ERK by U0126 suppressed CpG DNA-induced MMP-9 expression and its activity. CpG DNA stimulated $I{\kappa}B-{\alpha}$ degradation and luciferase activity. In addition, pretreatment of SN-50, the inhibitor of NF${\kappa}B$, strongly blocked the CpG DNA-induced MMP-9 expression and activity. Conclusion: These observations suggest that CpG DNA may play important roles in the activation of macrophages by regulating the production of MMP-9 via the sequential TLR9-ERK-NF${\kappa}B$ signaling pathway.

• Korean Journal of Microbiology
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• v.50 no.4
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• pp.296-301
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• 2014

Rotavirus is a major cause of acute gastroenteritis in young children in developed and developing countries. The use of probiotics for the treatment of gastrointestinal diseases is both safe and easily accessible. In this study, we evaluated the anti-rotaviral activities of probiotic mixtures in a Sprague-Dawley rat. 24 litters with their dams were randomly assigned to four groups; placebo, phosphate buffered saline (PBS), and two probiotic mixture (PRO-1 and PRO-2) groups. All rats were inoculated with rotavirus at dose of 8 log plaque forming units per rat at 5 days old. Animals in the PRO-1 and PRO-2 groups were orally administered probiotic mixtures 1 or 2, respectively, at a dose of 8 log colony forming units daily during 4 days. For control purposes, placebo and PBS groups were orally administered the same amount of placebo (containing maltose and polydextrose) or PBS once daily for 4 days, respectively. Antiviral analysis was performed by real-time quantitative PCR (RT-qPCR) and observing intestinal villi. As a result, weights of small intestines were greater in the PRO-1, PRO-2 groups than in control groups. Villi were short and villous epithelial necrosis was exhibited in control groups, but these morphological changes were not observed in PRO-1, PRO-2 treated rats. RT-qPCR analysis showed that VP7 gene level of rotavirus in fecal samples and small intestinal epithelial cells were lower in the PRO-1 and PRO-2 groups. These findings suggest that probiotic mixtures may be useful probiotics for the treatment of or as alternative therapies for rotaviral gastroenteritis.

• Asian-Australasian Journal of Animal Sciences
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• v.26 no.12
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• pp.1665-1671
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• 2013

Real-time quantitative PCR (qRT-PCR) is one of the important methods for investigating the changes in mRNA expression levels in cells and tissues. Selection of the proper reference genes is very important when calibrating the results of real-time quantitative PCR. Studies on the selection of reference genes in goat tissues are limited, despite the economic importance of their meat and dairy products. We used real-time quantitative PCR to detect the expression levels of eight reference gene candidates (18S, TBP, HMBS, YWHAZ, ACTB, HPRT1, GAPDH and EEF1A2) in ten tissues types sourced from Boer goats. The optimal reference gene combination was selected according to the results determined by geNorm, NormFinder and Bestkeeper software packages. The analyses showed that tissue is an important variability factor in genes expression stability. When all tissues were considered, 18S, TBP and HMBS is the optimal reference combination for calibrating quantitative PCR analysis of gene expression from goat tissues. Dividing data set by tissues, ACTB was the most stable in stomach, small intestine and ovary, 18S in heart and spleen, HMBS in uterus and lung, TBP in liver, HPRT1 in kidney and GAPDH in muscle. Overall, this study provided valuable information about the goat reference genes that can be used in order to perform a proper normalisation when relative quantification by qRT-PCR studies is undertaken.

• Journal of Life Science
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• v.29 no.6
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• pp.653-661
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• 2019

The first small interfering RNA (siRNA) therapeutics have recently been approved by the Food and Drug Administration in the U.S., and the demand for a new RNA therapeutics bioanalysis method-which is essential for pharmacokinetics, including the absorption, distribution, metabolism, and excretion of siRNA therapeutics-is rapidly increasing. The stem-loop real-time qPCR (RT-qPCR) assay is a useful molecular technique for the identification and quantification of small RNA (e.g., micro RNA and siRNA) and can be applied for the bioanalysis of siRNA therapeutics. When the anti-HPV E6/E7 siRNA therapeutic was used in preclinical trials, the established stem-loop RT-qPCR assay was validated. The limit of detection was sensitive up to 10 fM and the lower limit of quantification up to 100 fM. In fact, the reliability of the established test method was further validated in three intra assays. Here, the correlation coefficient of $R^2$>0.99, the slope of -3.10 ~ -3.40, and the recovery rate within ${\pm}20%$ of the siRNA standard curve confirm its excellent robustness. Finally, the circulation profiles of siRNAs were demonstrated in rat serum, and the pharmacokinetic properties of the anti-HPV E6/E7 siRNA therapeutic were characterized using a stem-loop RT-qPCR assay. Therefore, the stemloop RT-qPCR assay enables accurate, precise, and sensitive siRNA duplex quantification and is suitable for the quantification of small RNA therapeutics using small volumes of biological samples.

• Journal of Microbiology and Biotechnology
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• v.21 no.12
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• pp.1280-1286
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• 2011

A quantitative, real-time PCR method was developed to enumerate Lactobacillus plantarum IWBT B 188 during the malolactic fermentation (MLF) in Grauburgunder wine. The qRT-PCR was strain-specific, as it was based on primers targeting a plasmid DNA sequence, or it was L. plantarum-specific, as it targeted a chromosomally located plantaricin gene sequence. Two 50 l wine fermentations were prepared. One was inoculated with 15 g/hl Saccharomyces cerevisiae, followed by L. plantarum IWBT B 188 at $3.6{\times}10^6$ CFU/ml, whereas the other was not inoculated (control). Viable cell counts were performed for up to 25 days on MRS agar, and the same cells were enumerated by qRT-PCR with both the plasmid or chromosomally encoded gene primers. The L. plantarum strain survived under the harsh conditions in the wine fermentation at levels above $10^5$/ml for approx. 10 days, after which cell numbers decreased to levels of $10^3$ CFU/ml at day 25, and to below the detection limit after day 25. In the control, no lactic acid bacteria could be detected throughout the fermentation, with the exception of two sampling points where ca. $1{\times}10^2$ CFU/ml was detected. The minimum detection level for quantitative PCR in this study was $1{\times}10^2$ to $1{\times}10^3$ CFU/ml. The qRT-PCR results determined generally overestimated the plate count results by about 1 log unit, probably as a result of the presence of DNA from dead cells. Overall, qRT-PCR appeared to be well suited for specifically enumerating Lactobacillus plantarum starter cultures in the MLF in wine.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.64 no.4
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• pp.432-440
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• 2019

Global climate change exerts adverse effects on maize production. Among abiotic stresses, drought stress during the tasseling stage (VT) can increase anthesis-silking intervals (ASI) and decrease yield. We performed an evaluation of ASI and yield using a drought-sensitive line (Ki3) and a drought-tolerant line (Ki11) to analyze the correlation with ASI and yield. Moreover, the de novo data of Ki11 were analyzed to find putative novel transcripts related todrought stress in tropical maize. A total of 182 transcripts, with a log2 ratio >1.5, were found by comparing drought conditions to a control. The top 40 transcripts of high expression levels in the de novo analysis were selected and analyzed with PCR. Of the 40 transcripts, six novel transcripts were detected by quantitative real-time PCR (qRT-PCR) using seedling and VT stage samples. Five transcripts (transcripts_1, 12, 34, 35, and 40) were up-regulated in the Ki11 shoot at seedling stage, and transcripts_1, 12, and 40 were up-regulated at the re-watering stage after 12 h of drought stress. The transcripts_32 and 34 were up-regulated at the VT stage. Hence, transcript_34 possibly plays a significant role in drought tolerance during the seedling and VT stages. The transcript_32 was identified as chloramphenicol acetyltransferase (CAT) by Pfam domain analysis. The function of the other transcripts remained unknown. Further characterization of these novel transcripts in genetic regulation will be of great value for the improvement of maize production.

• Korean Journal of Food Science and Technology
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• v.49 no.2
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• pp.123-131
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• 2017

Norovirus causes frequent epidemic viral gastroenteritis in Korea. The team for the control of noroviral foodborne outbreaks (NOROTECL) executed a project to trace the cause of norovirus contamination in agricultural products and environmental samples to reduce norovirus outbreaks in Korea. Between January and November in 2015, the contaminations by norovirus and indicator microorganisms such as coliforms, Escherichia coil and male specific coliphage (MSC) were examined in 80 agricultural products, 80 soil samples, 78 human feces samples, 3 animal feces samples, 80 agricultural water samples and 80 river water samples. Semi-nested PCR and DNA sequencing revealed 18 genogroup I and 3 genogroup II noroviruses in a total of 18 samples. These noroviruses were validated by real-time (RT)-PCR analysis. For indicator microorganisms, coliform and E. coli were respectively detected in agricultural products (68, 1%), soils (88, 7%), human feces (44, 12.8%), animal feces (67, 67%), agricultural waters (74, 30%) and river waters (96, 51%). The MSC results revealed 14 positive samples.