• Journal of Life Science
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• v.17 no.7 s.87
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• pp.996-1001
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• 2007

To investigate whether sulindac, sulindac sulfone, and sulindac sulfide could affect cancer cell viabilities, human colorectal HCTl16 cells were treated with 10 ${\mu}M$ of each NSAID. Among treated NSAms, sulindac sulfide dramatically decreased the cell viabilities detected by MTS and the cytotoxic effect showed dose-dependent manner. To understand the molecular mechanism of cell death in response to sulindac sulfide treatment, we performed oligo DNA microarray analysis. We found that 23 genes were up-regulated more than 2 folds, whereas 33 genes were down-regulated more than 2 folds by treatment of 10 ${\mu}M$ sulindac sulfide. Among the up-regulated genes, we selected 3 genes (NAG-1, DDIT3, PCK2) and performed RT-PCR and quantitative real-time PCR to cofirm microarray data. The results of RT-PCR and real-time PCR were highly accorded with those of microarray experiment. As NAG-1 is well-known gene as tumor suppressor, we detected changes of NAG-1 expression by 10 ${\mu}M$ of sulindac, sulindac sulfone, and sulindac sulfide. The results of RT-PCR and quantitacve real-time PCR indicated that sulindac sulfide was the strongest inducer of NAG-1 among treated NSAIDS. This result implies that induction of NAG-1 by sulindac sulfide plays important role in cell death of colorectal cancer. Overall, we speculate that these results may be helpful in understanding the molecular mechanism of the cancer chemoprevention by sulindac sulfide in human colorectal cancer.

• Parasites, Hosts and Diseases
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• v.59 no.1
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• pp.77-82
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• 2021

As malaria remains a major health problem worldwide, various diagnostic tests have been developed, including microscopy-based and rapid diagnostic tests. LabChip real-time PCR (LRP) is a small and portable device used to diagnose malaria using lab-on-a-chip technology. This study aimed to evaluate the diagnostic performance of LRP for detecting malaria parasites. Two hundred thirteen patients and 150 healthy individuals were enrolled from May 2009 to October 2015. A diagnostic detectability of LRP for malaria parasites was compared to that of conventional RT-PCR. Sensitivity of LRP for Plasmodium vivax, P. falciparum, P. malariae, and P. ovale was 95.5%, 96.0%, 100%, and 100%, respectively. Specificity of LRP for P. vivax, P. falciparum, P. malariae, and P. ovale was 100%, 99.3%, 100%, and 100%, respectively. Cohen's Kappa coefficients between LRP and CFX96 for detecting P. vivax, P. falciparum, P. malariae, and P. ovale were 0.96, 0.98, 1.00, and 1.00, respectively. Significant difference was not observed between the results of LRP and conventional RT-PCR and microscopic examination. A time required to amplify DNAs using LRP and conventional RT-PCR was 27 min and 86 min, respectively. LRP amplified DNAs 2 times more fast than conventional RT-PCR due to the faster heat transfer. Therefore, LRP could be employed as a useful tool for detecting malaria parasites in clinical laboratories.

• Korean Journal of Veterinary Service
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• v.44 no.3
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• pp.163-168
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• 2021

The rapid and reliable detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays a key role in isolating infected patients and preventing further viral transmission. In this study, we evaluated the clinical diagnostic performances of a commercial real-time reverse transcription loop-mediated isothermal amplification (RRT-LAMP) assay (Isopollo^® COVID-2 assay, M-monitor, Daegu, Korea) using eighty COVID-19 suspected clinical samples and compared these with the results of a commercial real-time reverse transcription polymerase chain reaction (RT-qPCR) assay (Allplex^TM 2019-nCoV rRT-QPCR Assay, SeeGene, Seoul, Korea). The results of the RRT-LAMP assay targeting the N or RdRp gene of SARS-CoV-2 showed perfect agreement with the RT-qPCR assay results in terms of detection. Furthermore, the RRT-LAMP assay was completed in just within a 20-min reaction time, which is significantly faster than about the 2 h currently required for the RT-qPCR assay, thus enabling prompt decision making regarding the isolation of infected patients. The RRT-LAMP assay will be a valuable tool for rapid, sensitive, and specific detection of SARS-CoV-2 in human or unexpected animal clinical cases.

• Biomedical Science Letters
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• v.22 no.4
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• pp.215-219
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• 2016

The problems of tuberculosis and its drug resistance are very severe. Therefore, rapid and accurate drug susceptibility assay is required. Recently, there has been an increased understanding of the genetic mechanism of Mycobacterium tuberculosis (MTB) drug resistance as well as advancement of molecular technologies. While many gene mutations correlate well with drug resistance, many genes do not show a strong correlation with drug resistance. For this reason, the current study assessed the utility of rpoB mRNA as a target to detect live mycobacteria. In this study, RT-PCR targeting of rpoB mRNA in BCG treated with rifampin was performed. Conventional RT-PCR and real-time PCR targeting rpoB mRNA as well as 85B mRNA was performed to determine whether these two methods could distinguish between viable and non-viable MTB. The levels of rpoB and 85B mRNA detected by RT- PCR were compared in parallel with colony forming unit counts of BCG that were treated with rifampin for different periods of time. The data suggests that that even though both mRNA levels of rpoB and 85B decreased gradually when rifampin-treatment increased, the rpoB mRNA seemed to represent live bacteria better than 85B mRNA. This study clearly indicates that RT-PCR is a good method to monitor viable cell counts in the liquid culture treated with the anti-tuberculosis drug.

• Korean Journal of Veterinary Service
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• v.38 no.2
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• pp.107-116
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• 2015

In this study, we developed a rapid, sensitive and specific reverse-transcriptase loop-mediated isothermal amplification (RT-LAMP) assay for detection of swine influenza viruse (SIV) including major subtypes of swine influenza viruses H1N1, H1N2 and H3N2, and a novel subtype of influenza A virus that accidentally infected in pig population. The RT-LAMP was completed in 40 min at $58^{\circ}C$ and the sensitivity of the RT-LAMP ($1copy/{\mu}L$) was 10-fold higher than conventional reverse transcription-polymerase chain reaction (RT-PCR) ($10copy/{\mu}L$) and the same to real time RT-PCR ($1copy/{\mu}L$). Also, the result of the RT-LAMP can be confirmed without any detection system. Therefore, the RT-LAMP could be a alternative diagnostic method for SIV detection in national SIV monitoring system and clinical diagnostic laboratory in the future.

• Journal of Microbiology and Biotechnology
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• v.23 no.5
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• pp.630-636
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• 2013

Recently, there has been a growing body of evidence showing that cellular microRNAs (miRNAs) are involved in virus-host interactions. Numerous studies have focused on analyses of the expression profiles of cellular miRNAs, but the expression patterns of Dicer, which is responsible for the generation of miRNAs, have only rarely been explored in Gallus gallus. We developed a duplex real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay for the relative quantification of the mRNAs of Dicer and ${\beta}$-actin in G. gallus. To apply this method, the expression of Dicer in avian cells after infection with avian leukosis virus subgroup J (ALV-J) was detected using our established duplex real-time RT-PCR. The duplex real-time RT-PCR assay is sufficiently sensitive, specific, accurate, reproducible, and cost-effective for the detection of Dicer in G. gallus. Furthermore, this study, for the first time, demonstrated that ALV-J can induce differential expression of Dicer mRNA in the ALV-J-infected cells.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.12
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• pp.7621-7628
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• 2013

Human epidermal growth factor receptor (HER) status is an important prognostic factor in breast cancer. There is no globally accepted method for determining its status, and which method is most precise is still a matter of debate. We here analyzed HER2 mRNA expression by quantitative reverse transcription-PCR (qRT-PCR) and HER2 DNA amplification using multiplex ligation-dependent probe amplification (MLPA). In parallel, we performed a routine evaluation of HER2 protein by immunohistochemistry (IHC). To assess the accuracy of the RT-PCR and MLPA techniques, a combination of IHC and fluorescence in situ hybridization (FISH) was used, substituting FISH when the results of IHC were ambiguous (2+) and for those IHC results that disagreed with MLPA and qRT-PCR, this approach being termed IHC-FISH. The IHC results for four samples were not compatible with the MLPA and qRT-PCR results; the MLPA and qRT-PCR results for these samples were confirmed by FISH. The correlations between IHC-FISH and qRT-PCR or MLPA were 0.945 and 0.973, respectively. The ASCO/CAP guideline IHC/FISH correlation with MLPA was (0.827) and with RT-PCR was (0.854). The correlations between the IHC results (0, 1+ as negative, and 3+ as positive) and qRT-PCR and MLPA techniques were 0.743 and 0.831, respectively. Given the shortcomings of IHC analysis and greater correlations between MLPA, qRT-PCR, and FISH methods than IHC analysis alone with each of these three methods, we propose that MLPA and real-time PCR are good alternatives to IHC. However a suitable cut-off point for qRTPCR is a prerequisite for determining the exact status of HER2.

• Molecular & Cellular Toxicology
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• v.4 no.3
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• pp.211-217
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• 2008

To clarify the DNA damage from reactive oxygen species, we measured the DNA damage through Fpg/Endo III FLARE (Fragment Length Analysis with Repair Enzyme) assay and real time RT-PCR. The 80 SD rats assigned to 4 dose groups exposed to cumene vapor for 90 days. With Fpg/Endo III FLARE assay in hepatocytes, we found the OTM (Olive Tail Moment) and TL (Tail Length) significantly increased in no-enzyme treated and Fpg-treated control and 8 ppm groups with 28 days exposure. In Endo III-treated 8 ppm group, significantly increased the values with 90 days exposure. With lymphocytes, it was founded the values significantly increased in no-enzyme treated 800 ppm group in 28 and 90 days. It was significantly increased in Endo III-treated 80 ppm for 28 days and 800 ppm for 90 days. From the above findings, FLARE assay was suggested as being available as a biological marker for DNA damage induced by cumene exposure in SD rats. And we used real time RT-PCR for the OGG1 mRNA expression, it had dose-dependent biologic effects in 1 day exposure, but decrease the levels of rOGG1 mRNA. Our findings provide evidence that cumene exposure may cause suppression of rOGG1 in the rat hepatocytes or lymphocytes.

• Journal of Microbiology and Biotechnology
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• v.18 no.4
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• pp.648-657
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• 2008

In this study, we used functional genomic strategies, proteomics and quantitative real-time (qRT)-PCR, to advance our understanding of genes associated with fumonisin production in the fungus Fusarium verticillioides. Earlier studies have demonstrated that deletion of the FCC1 gene, which encodes a C-type cyclin, leads to a drastic reduction in fumonisin production and conidiation in the mutant strain (FT536). The premise of our research was that comparative analysis of F. verticillioides wild-type and FT536 proteomes will reveal putative proteins, and ultimately corresponding genes, that are important for fumonisin biosynthesis. We isolated proteins that were significantly upregulated in either the wild type or FT536 via two-dimensional polyacrylamide gel electrophoresis, and subsequently obtained sequences by mass spectrometry. Homologs of identified proteins, e.g., carboxypeptidase, laccase, and nitrogen metabolite repression protein, are known to have functions involved in fungal secondary metabolism and development. We also identified gene sequences corresponding to the selected proteins and investigated their transcriptional profiles via quantitative real-time (qRT)-PCR in order to identify genes that show concomitant expression patterns during fumonisin biosynthesis. These genes can be selected as targets for functional analysis to further verify their roles in $FB_1$ biosynthesis.

• Korean Journal of Veterinary Service
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• v.41 no.1
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• pp.29-39
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• 2018

In this study, we developed a sensitive and specific reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for rapid visual detection of foot-and-mouth disease virus (FMDV) circulated in Korea. The RT-LAMP was completed in 40 min at $62^{\circ}C$ and the results of the assay were directly detected by naked eye without any detection process. The assay specifically amplified all 7 serotypes of FMDV RNAs but not amplified other viral and cellular nucleic acids. The sensitivity of the RT-LAMP was $10^2$, $10^3$ and $10^3TCID_{50}/mL$ for serotype O, A and Asia 1 FMDV, respectively, which was comparable to conventional reverse transcription polymerase chain reaction (RT-PCR) and relatively lower than that of real time quantitative RT-PCR (qRT-PCR). Clinical evaluation of the RT-LAMP using different serotypes of Korean and foreign FMDV strains showed a 100% (35/35) agreement with the results of the RT-PCR and qRT-PCR. These results indicated that RT-LAMP assay developed in this study could be a valuable diagnostic method for FMDV monitoring and surveillance.