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

• Research in Plant Disease
• /
• v.22 no.3
• /
• pp.178-183
• /
• 2016

Soybean yellow mottle mosaic virus (SYMMV) is a new emerging plant virus detected in soybean (Glycine max) in Korea. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for rapid detection of SYMMV has been developed. In this study, we have designed primers (SYMM-F3/B3/FIP/BIP) specific to sequences from the coat protein gene of SYMMV genome. Sensitivity analysis showed that RT-LAMP was 10 to 100 times more sensitive than reverse transcription polymerase chain reaction (RT-PCR). The optimal reaction condition of RT-LAMP was determined at $65^{\circ}C$ for 50 minutes. The result indicates that RT-LAMP assay does not require special equipment and long time for SYMMV detection. Therefore, it can be an alternative detection method of RT-PCR in laboratory.

• Journal of Environmental Health Sciences
• /
• v.34 no.2
• /
• pp.170-174
• /
• 2008

The strains of Salmonella othmarschen and Norovirus isolated from outbreak A, the funeral ceremonies incidents in Ouri-shi of Oyeonggido in, 2007, were subjected to genotyping by using PFGE and extraction of RNA by using RT-PCR. 24 Salmonella othmarschen were isolated from 37 cases (patients 12 cases, cookers 4 cases, foods 7cases, knife, kitchen board, dish towel 4 cases, etc) and PFGE patterns showed that 22 strains isolated from the same incident were almost of the same pattern and 2 strains exhibited 50% of the pattern. Antimicrobial susceptibility patterns were such that there were 22 case susceptibility patterns and 2 case resistance patterns (strain 4: AM, CF, CIP, NA, TE TIC and strain 13: CF, CZ). Norovirus G1 (1case), Norovirus G2 (7cases), Norovirus G1,G2 (1case) were detected by using RT-PCR. The fine typing ability and reliability of PFGE and BioNumerics are helpful for establishing out the foodborne pathogens of outbreaks.

• Research in Plant Disease
• /
• v.10 no.1
• /
• pp.39-43
• /
• 2004

Recent occurrence of virus diseases on watermelon plants cultivated in Jeonnam province was investigated from 1998 to 2002. While virus diseases were severely occurred on watermelon cultivated in green house in 1998, those of open field were severer than in green house since 2000. When 128 samples collected from different fields were examined by electron microscopy, 87.8％ of the samples contained rod-shaped or filamentous virus particles. RT-PCR analysis of the samples revealed that Cucumber green mottle mosaic virus (CGMMV) was only detected from collected samples at May. Watermelon mosaic virus (WMV) was most frequently found and CGMMV and Zucchini yellow mosaic virus (ZYMV) were slightly at June and July. However Cucumber mosaic virus (CMV) and Papaya ringspot virus (PRSV) have not been detected.

• Microbiology and Biotechnology Letters
• /
• v.49 no.3
• /
• pp.440-448
• /
• 2021

Helicobacter pylori (H. pylori) establishes long-term infections associated with severe gastric diseases such as peptic ulceration and gastric cancer. Exposure to an antibacterial agent can help regulate the expression levels of its pathogenic genes. In this study, we analyzed the transcriptional changes in H. pylori genes induced by β-caryophyllene. We used next-generation sequencing (NGS) to analyze RNA expression changes, and reverse transcription-polymerase chain reaction (RT-PCR) was performed as required to verify the results. The NGS results showed that 30 out of 1,632 genes were expressed differentially by β-caryophyllene treatment. Eleven genes associated with DNA replication, virulence factors, and T4SS components were significantly downregulated. RT-PCR confirmed that treatment reduced the expression levels of 11 genes. RT-PCR showed the reduced expression of 11 genes (dnaE, dnaN, holB, gyrA, cagA, vacA, secA, flgE, virB2, virB4, and virB8) following β-caryophyllene treatment. These results suggest that β-caryophyllene can modulate various H. pylori pathogenic determinants and be a potential therapeutic agent for H. pylori infection.

• Korean Journal of Agricultural Science
• /
• v.43 no.1
• /
• pp.21-27
• /
• 2016

Apple stem grooving virus (ASGV), Apple chlorotic leaf spot virus (ACLSV), and Apple stem pitting virus (ASPV) have been known to induce top working disease causing economical damage in apple. Occurrences of these three viruses in pome fruit trees, including apple, have been reported around the world. The transmission of the three viruses was reported by grafting, and there was no report of transmission through mechanical contact, insect vector, or seed except some herbaceous hosts of ASGV. As RNA extraction methods for fruit trees, Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) and multiplex RT-PCR techniques have been improved for reliability and stability, and low titer viruses that could not be detected in the past have become detectable. We studied the seed transmission ability of three apple viruses through apple seedling diagnosis using RT-PCR. Nineteen seeds obtained from commercially grown apple were germinated and two of the resulting plants were ASGV positive. Seven clones of the amplified ASGV coat protein (CP) genes of these isolates were sequenced. Overall sequence identities were 99.84% (nucleotide) and 99.76% (amino acid). Presence of a previously unreported single nucleotide and amino acid variation conserved in all of these clones suggests a possible association with seed transmission of these 'S' isolates. A phylogenetic tree constructed using ASGV CP nucleotide sequences showed that isolate S sequences were grouped with Korean, Chinese, Indian isolates from apple and Indian isolates from kiwi.

• Korean Journal of Animal Reproduction
• /
• v.19 no.1
• /
• pp.55-63
• /
• 1995

The putative totipotency germ cells has a relative abundance of alkaline phosphatases. Thus, histological staining of AP activity offers a new route to isolate totipotent cells and also provides insights into culture systems of these cells. Furthermore, the AP staining technique is simple and fast, requires only the napthol AS/MS substrate in combination with trapping diazonium salts such as fast red or fast blue. However, our unexpected finding was that AP staining of mouse ES cells were detected in the undifferentiaed epiblast-derived cells as well as several types of differentiating cells. This findings are different from results of Talbot et al. (1993) reported usefulness of the AP staining and implies that histological staining of AP may not by useful to determine undifferentiaed state or totipotency of ES cells. Thus, we have investigated the patterns of AP expression by RT-PCR in order to identify a marker of undifferentiated ES/primordial germ (PG) cells. In RT-PCR analysis, embryonic (E)-AP was detected only in undifferentiated ES cells, but intestinal(I)-AP was not detected in all of the examined ES and PG cells. In addition, nonspecific (NS)-AP wasdetected in undifferentiated PG cell from day 7, 5 to 13 of gestation. Histological activity of AP in ES cells was completely suppressed by addition of L-phenylalanine (Phe), L-homoarginine (Har), and L-phenylalanylglycylglycine (PheGlyGly) as an inhibitor, but RT-PCR showed the same results as in the absence of an inhibitors. Our findings suggested that expression of E-AP and NS-AP may use as a marker to determine the undifferentiated status in ES and PG cells.

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

• Asian-Australasian Journal of Animal Sciences
• /
• v.18 no.6
• /
• pp.892-897
• /
• 2005

Macrophage colony-stimulating factor (M-CSF) is a growth factor required for growth and differentiation of mononuclear phagocyte lineage. Total and 16 poly (A) mRNA of bovine M-CSF were isolated from healthy bovine peripheral mononuclear cells stimulated by phobol 12-myristste 13-acetate (TPA). The more compatible cultured mononuclear cells were 5${\times}$10/ml for RNA isolation. TPA-activated mononuclear cells increased the level of M-CSF-mRNA more than concanavalin A (Con A) and lipopolysaccharide (LPS). The optimal analysis of reverse transcriptase-polymerase chain reaction (RT-PCR) for14 Macrophage colonystimulating factor (M-CSF) as a growth factor required for bovine M-CSF was denaturation at 94$^{\circ}C$ for 1 minute, annealing at 57$^{\circ}C$ for 1 minute, extension at 72$^{\circ}C$ for 1 minute for 30 cycles. The size of cDNA of bovine M-CSF by RT-PCR was 774 base pairs. A 774 base pairs cDNA encoding bovine M-CSF was synthesized by reverse transcriptase polymerase chain reaction (RT-PCR). Ligated cDNA was transformed to competent cells and then plasmid isolation and digestion was performed. Molecular cloning and sequencing were performed for cDNA of bovine M-CSF. The size of cloned cDNA of bovine M-CSF was 774base pairs. The homology of base sequence and amino acid sequence was 88% and 86% compared with known human M-CSF, respectively. From a high degree of sequence similarity, the obtained cDNA of bovine M-CSF is thought be a specific gene of bovine M-CSF.

• Journal of Animal Reproduction and Biotechnology
• /
• v.34 no.4
• /
• pp.280-291
• /
• 2019

Potential utility of 14 candidate housekeeping genes as normalization reference for RT-qPCR analysis with developmental samples (fertilized eggs to late veliger larvae) in Pacific abalone Haliotis discus hannai was evaluated using four different statistical algorithms (geNorm, NormFinder, BestKeeper and comparative ΔCT method). Different algorithms identified different genes as the best candidates, and geometric mean-based final ranking from the most to the least stable expression was as follow: RPL5, RPL4, RPS18, RPL8, RPL7, UBE2, RPL7A, GAPDH, RPL36, PPIB, EF1A, ACTB and B-TU. The findings were further validated via relative quantification of metallothionein (MT) transcripts using the stable and unstable reference genes, and expression levels of MT were greatly influenced according to the choice of reference genes. In overall, our data suggest that RPL5 and RPS18, either singly or in combination, are appropriate for normalizing gene expression in developmental samples of this abalone species, whereas ACTB, B-TU and EF1A are less stable and not recommended. In addition, our findings propose that standard deviations in geometric ranking as well as geometric mean itself should also be taken into account for the final selection of reference gene(s). This study could be a useful basis to facilitate the generation of accurate and reliable RT-qPCR data with developmental samples in this abalone species.