• Asian-Australasian Journal of Animal Sciences
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• 제28권11호
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• pp.1649-1656
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• 2015

Gene expression profiling has offered new insights into postmortem molecular changes associated with meat quality. To acquire reliable transcript quantification, high quality RNA is required. The objective of this study was to analyze integrity of RNA isolated from chicken skeletal muscle (pectoralis major) and its capability of serving as the template in quantitative real-time polymerase chain reaction (qPCR) as a function of postmortem intervals representing the end-points of evisceration, carcass chilling and aging stages in chicken abattoirs. Chicken breast muscle was dissected from the carcasses (n = 6) immediately after evisceration, and one-third of each sample was instantly snap-frozen and labeled as 20 min postmortem. The remaining muscle was stored on ice until the next rounds of sample collection (1.5 h and 6 h postmortem). The delayed postmortem duration did not significantly affect $A_{260}/A_{280}$ and $A_{260}/A_{230}$ ($p{\geq}0.05$), suggesting no altered purity of total RNA. Apart from a slight decrease in the 28s:18s ribosomal RNA ratio in 1.5 h samples (p<0.05), the value was not statistically different between 20 min and 6 h samples ($p{\geq}0.05$), indicating intact total RNA up to 6 h. Abundance of reference genes encoding beta-actin (ACTB), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), hypoxanthine-guanine phosphoribosyltransferase (HPRT), peptidylprolylisomerase A (PPIA) and TATA box-binding protein (TBP) as well as meat-quality associated genes (insulin-like growth factor 1 (IGF1), pyruvate dehydrogenase kinase isozyme 4 (PDK4), and peroxisome proliferator-activated receptor delta (PPARD) were investigated using qPCR. Transcript abundances of ACTB, GAPDH, HPRT, and PPIA were significantly different among all postmortem time points (p<0.05). Transcript levels of PDK4 and PPARD were significantly reduced in the 6 h samples (p<0.05). The findings suggest an adverse effect of a prolonged postmortem duration on reliability of transcript quantification in chicken skeletal muscle. For the best RNA quality, chicken skeletal muscle should be immediately collected after evisceration or within 20 min postmortem, and rapidly preserved by deep freezing.

• Asian-Australasian Journal of Animal Sciences
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• 제33권12호
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• pp.2021-2030
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• 2020

Objective: Quantitative polymerase chain reaction (qPCR) has been extensively used in the field of mesenchymal stem cell (MSC) research to elucidate their characteristics and clinical potential by normalization of target genes against reference genes (RGs), which are believed to be stably expressed irrespective of various experimental conditions. However, the expression of RGs is also variable depending on the experimental conditions, which may lead to false or contradictory conclusions upon normalization. Due to the current lack of information for a clear list of stable RGs in bovine MSCs, we conducted this study to identify suitable RGs in bovine MSCs. Methods: The cycle threshold values of ten traditionally used RGs (18S ribosomal RNA [18S], beta-2-microglobulin [B2M], H2A histone family, member Z [H2A], peptidylprolyl isomerase A [PPIA], ribosomal protein 4 [RPL4], succinate dehydrogenase complex, subunit A [SDHA], beta actin [ACTB], glyceraldehyde-3-phosphate dehydrogenase [GAPDH], TATA box binding protein [TBP], and hypoxanthine phosphoribosyltrasnfrase1 [HPRT1]) in bovine bone marrow-derived MSCs (bBMMSCs) were validated for their stabilities using three types of RG evaluation algorithms (geNorm, Normfinder, and Bestkeeper). The effect of validated RGs was then verified by normalization of lineage-specific genes (fatty acid binding protein 4 [FABP4] and osteonectin [ON]) expressions during differentiations of bBMMSCs or POU class 5 homeobox 1 (OCT4) expression between bBMMSCs and dermal skins. Results: Based on the results obtained for the three most stable RGs from geNorm (TBP, RPL4, and H2A), Normfinder (TBP, RPL4, and SDHA), and Bestkeeper (TBP, RPL4, and SDHA), it was comprehensively determined that TBP and RPL4 were the most stable RGs in bBMMSCs. However, traditional RGs were suggested to be the least stable (18S) or moderately stable (GAPDH and ACTB) in bBMMSCs. Normalization of FABP4 or ON against TBP, RPL4, and 18S presented significant differences during differentiation of bBMMSCs. However, although significantly low expression of OCT4 was detected in dermal skins compared to that in bBMMSCs when TBP and RPL4 were used in normalization, normalization against 18S exhibited no significance. Conclusion: This study proposes that TBP and RPL4 were suitable as stable RGs for qPCR study in bovine MSCs.

• The Plant Pathology Journal
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• 제39권4호
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• pp.384-396
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• 2023

Colletotrichum acutatum species complex is one of the most important groups in the genus Colletotrichum with a high species diversity and a wide range of host plants. C. acutatum and related species have been collected from different plants and locations in Korea and deposited into the Korean Agricultural Culture Collection (KACC), National Institute of Agricultural Sciences since the 1990s. These fungal isolates were previously identified based mainly on morphological characteristics, and a limitation of molecular data was provided. To confirm the identification of species, 64 C. acutatum species complex isolates in KACC were used in this study for DNA sequence analyses of six loci: nuclear ribosomal internal transcribed spacers (ITS), betatubulin 2 (TUB2), histone-3 (HIS3), glyceraldehyde3-phosphate dehydrogenase (GAPDH), chitin synthase 1 (CHS-1), and actin (ACT). The molecular analysis revealed that they were identified in six different species of C. fioriniae (24 isolates), C. nymphaeae (21 isolates), C. scovillei (12 isolates), C. chrysanthemi (three isolates), C. lupini (two isolates), and C. godetiae (one isolate), and a novel species candidate. We compared the hosts of KACC isolates with "The List of Plant Diseases in Korea", previous reports in Korea and global reports and found that 23 combinations between hosts and pathogens could be newly reported in Korea after pathogenicity tests, and 12 of these have not been recorded in the world.

• Parasites, Hosts and Diseases
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• 제54권1호
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• pp.39-46
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• 2016

Theileria annulata is a tick-borne intracellular protozoan parasite that causes tropical theileriosis, a fatal bovine lymphoproliferative disease. The parasite predominantly invades bovine B lymphocytes and macrophages and induces host cell transformation by a mechanism that is not fully comprehended. Analysis of signaling pathways by quantitative real-time PCR (qPCR) could be a highly efficient means to understand this transformation mechanism. However, accurate analysis of qPCR data relies on selection of appropriate reference genes for normalization, yet few papers on T. annulata contain evidence of reference gene validation. We therefore used the geNorm and NormFinder programs to evaluate the stability of 5 candidate reference genes; 18S rRNA, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ACTB (${\beta}-actin$), PRKG1 (protein kinase cGMP-dependent, type I) and TATA box binding protein (TBP). The results showed that 18S rRNA was the reference gene most stably expressed in bovine PBMCs transformed and non-transformed with T. annulata, followed by GAPDH and TBP. While 18S rRNA and GAPDH were the best combination, these 2 genes were chosen as references to study signaling pathways involved in the transformation mechanism of T. annulata.

• 한국균학회지
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• 제44권3호
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• pp.201-205
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• 2016

2015년 8월경 강원도 영월군의 고려엉겅퀴 재배 농가에서 잎에 소형 점무늬 증상이 발생하였다. 초기에는 고려엉겅퀴 잎에 회갈색 내지 갈색의 작은 점이 나타나며, 병이 진전되면 부정형의 진한 갈색의 병반으로 커지면서 크게 확대되고 결국 잎 전체가 진한 갈색 내지 흑색으로 변하였다. 병든 잎에서 Stemphylium균이 분리되었으며, 분생포자 크기와 분생포자경의 길이 등을 고려한 균학적 특성에 의해 Stemphylium lycopersici 혹은 Stemphylium solani, Stemphylium xanthosomatis와 유사한 것으로 나타났다. Ribosomal internal transcribed spacer (ITS) 영역, elongation factor 1 alpha (EF-$1{\alpha}$), glyceraldehydes-3-phosphate dehydrogenase (gpd), vacuolar membrane ATPase catalytic subunit A gene (vmaA)와 vacuolar biogenesis gene (vpsA) 사이의 noncoding 영역을 대상으로 한 다자위 염기서열 분석에 의해 분리균은 모두 S. lycopersici로 확인되었다. 고려엉겅퀴 잎을 대상으로 상처구와 무상처구로 나누어 분리균의 포자 현탁액을 접종하여 병원성을 확인한 결과, 접종 3일 후부터 상처의 유무에 관계없이 분리균을 접종한 부위에서 병반이 관찰되었다. 따라서 본 결과를 토대로 본 병을 S. lycopersici에 의한 고려엉겅퀴 점무늬병으로 명명하며, 고려엉겅퀴 점무늬병의 발생을 국내 최초로 보고한다.

• Fisheries and Aquatic Sciences
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• 제22권12호
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• pp.28.1-28.8
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• 2019

Background: In the present study, we evaluated four commonly used housekeeping genes, viz., actin-β, elongation factor-1α (EF1α), acidic ribosomal protein (ARP), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as internal references for quantitative analysis of immune genes in nervous necrosis virus (NNV)-infected seven-band grouper, Hyporthodus septemfasciatus. Methods: Expression profiles of the four genes were estimated in 12 tissues of healthy and infected seven-band grouper. Expression stability of the genes was calculated using the delta Ct method, BestKeeper, NormFinder, and geNorm algorithms. Consensus ranking was performed using RefFinder, and statistical analysis was done using GraphpadPrism 5.0. Results: Tissue-specific variations were observed in the four tested housekeeping genes of healthy and NNV-infected seven-band grouper. Fold change calculation for interferon-1 and Mx expression using the four housekeeping genes as internal references presented varied profiles for each tissue. EF1α and actin-β was the most stable expressed gene in tissues of healthy and NNV-infected seven-band grouper, respectively. Consensus ranking using RefFinder suggested EF1α as the least variable and highly stable gene in the healthy and infected animals. Conclusions: These results suggest that EF1α can be a fairly better internal reference in comparison to other tested genes in this study during the NNV infection process. This forms the pilot study on the validation of reference genes in Hyporthodus septemfasciatus, in the context of NNV infection.

• 대한소아치과학회지
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• 제45권2호
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• pp.242-249
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• 2018

이 연구의 목적은 정량적 실시간 중합효소 연쇄 반응법을 이용하여 발거된 과잉치 치수 및 치주인대 줄기세포의 유전자 발현을 비교하는 것이다. 전신병력이 없는 만 6세 남아 2명의 상악 전치부에 매복된 과잉치를 발거하였다. 같은 날 치수 및 치주인대 세포를 채취하였고, 3계대까지 계대배양하였다. 분석을 위해 상아질모세포 특이 유전자인 Alkaline phosphatase (ALP), Dentin Matrix Protein 1 (DMP-1), Dentin sialophosphoprotein (DSPP), Osteocalcin (OCN) 그리고 Osteonectin (ONT)을 사용했고, glyceraldehyde 3-phosphate dehydrogenase (GAPDH)을 대조군으로 설정했다. 과잉치 치수 세포에서는 ONT, OCN, ALP, DMP-1, DSPP 순서로 발현량이 많았고, 치주인대 세포에서는 DMP-1과 DSPP의 순서만 바뀌었다. 치주인대 세포보다 치수 세포에서 모든 유전자의 발현량이 많았다. 이러한 상아질모세포의 특성을 고려해 보았을 때, 다른 조직으로의 분화 가능성이 있는 과잉치 줄기세포는 유용한 공여부로서 그 잠재력이 있음을 알 수 있었다.

• Journal of Microbiology and Biotechnology
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• 제16권12호
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• pp.1904-1911
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• 2006

The cell-free extracts of 250 yeasts were screened for their in vitro anti-angiogenic activity, to develop a new cancer metastasis inhibitor. Saccharomyces cerevisiae K-7 was selected as the producer of the anti-angiogenic agent, because it had the highest anti-angiogenic activity. The anti-angiogenic agent was produced maximally from hydrolysates of Saccharomyces cerevisiae K-7, when the yeast was cultured in yeast extract-peptone-dextrose medium at 30$^{\circ}C$ for 24 h, and cell-free extracts were than digested with pepsin for 4 h at 37$^{\circ}C$. The anti-angiogenic agent was further purified by ultrafiltration, Sephadex G-25 gel permeation chromatography and reverse-phase HPLC, and the anti-angiogenic activity of the final purified preparation was 72.7% at 10 $\mu$M/egg. The purified anti-angiogenic agent was found to originate from the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) molecule of Saccharomyces cerevisiae K-7, and its peptide sequence was Val-Ser-Trp-Tyr-Asp-Asn-Glu-Tyr-Gly-Tyr-Ser-Thr-Arg-Val-Val-Asp. In the MTT assay, the shape of the HT-l 080 cell was clearly changed to a circular type at 0.2 mM purified anti-angiogenic agent. This result indicated that the growth of the HT-I080 cell was significantly inhibited at 0.2 mM of the purified anti-angiogenic agent. The MMP activity of the treated HT-l080 cells was not affected, evidenced by the gelatin zymography, indicating that the anti-angiogenic mechanism of the purified anti-angiogenic agent is not mediated through MMP activity.

• Asian Pacific Journal of Cancer Prevention
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• 제14권8호
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• pp.4883-4889
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• 2013

Background: The most common incident cancer and cause of cancer-related deaths in women is breast cancer. The Myc gene is upregulated in many cancer types including breast cancer, and it is considered as a potential anti-cancer drug target. The present study was conducted to evaluate the Myc (gene and protein) expression pattern in an experimental mammary tumour model in rats. Materials and Methods: Thirty six Sprague Dawley rats were divided into: Experimental group (26 animals), which received the chemical carcinogen N-methyl nitrosourea (MNU) and a control group (10 animals), which received vehicle only. c-Myc oncoprotein and its mRNA expression pattern were evaluated using immunohistochemistry (IHC) and semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR), respectively, in normal rat mammary tissue and mammary tumours. The rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as internal control for semi-quantitative RT-PCR. Results: Histopathological examination of mammary tissues and tumours from MNU treated animals revealed the presence of premalignant lesions, benign tumours, in situ carcinomas and invasive carcinomas. Immunohistochemical evaluation of tumour tissues showed upregulation and heterogeneous cellular localization of c-Myc oncoprotein. The expression levels of c-Myc oncoprotein were significantly elevated (75-91%) in all the tumours. Semi-quantitative RT-PCR revealed increased expression of c-Myc mRNA in mammary tumours compared to normal mammary tissues. Conclusions: Further large-scale investigation study is needed to adopt this experimental rat mammary tumour model as an in vivo model to study anti-cancer strategies directed against Myc or its downstream partners at the transcriptional or post-transcriptional level.

• 대한의생명과학회지
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• 제29권1호
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• pp.48-52
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• 2023

Formaldehyde use is associated with serious health risks, which can affect medical personnel and technicians. Therefore, we investigated the efficacy of an alternative fixative, with respect to two types of formalin fixatives, by hematoxylin and eosin (H&E) staining, periodic acid Schiff (PAS) staining, immunohistochemical (IHC) staining, and RNA extraction. For H&E staining, the circular nucleus was stained dark blue by the basic dye hematoxylin and the cytoplasm was stained red by the acid dye eosin in all three fixative samples. No difference was found in the Duksan General Science (DGS), Sigma-Aldrich, and Core-Fix fixative samples (Corebiotech) used to fix kidney tissue, after PAS staining. IHC staining showed that CD4 was significantly increased in the lippolysaccharide (LPS)-treated group compared to the control group (vehicle), confirming the changes in specific molecules. The quantity and quality of RNA from tissues fixed in the three types of fixatives were evaluated. The average concentration of RNA was 106 ng/µL and average purity at A 260/280 ratio was 1.7~2.0, regardless of fixative used. For quality of protein, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein was confirmed by Western blotting. In conclusion, Core-Fix can be used as a fixative for pathological tissues, in histological and molecular diagnoses.