• 제목/요약/키워드: mRT-PCR

검색결과 2,049건 처리시간 0.033초

해녀콩(Canavalia lineata THUNB. DC.) 추출물의 멜라닌 생성 억제 효과 (Anti-melanogenesis Effect of Canavalia lineata Extract)

  • 부희정;류기중;이선주
    • 대한화장품학회지
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    • 제30권4호
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    • pp.485-489
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    • 2004
  • 피부에서 melanin은 자외선 차단의 주요한 역할을 한다. Tyrosinase는 멜라닌 생합성과정에서 초기 단계에 관여하는 중요한 효소로서 이것의 조절을 통한 피부 멜라닌화 억제에 관해 많은 연구가 되어져왔다. 본 연구에서는 해녀콩 추출물에서 mushroom tyrosinase 활성억제, B16F10 melanoma 세포를 이용한 dopa oxidase 활성억제 및 멜라닌 합성 억제 효과를 확인하였다. Tyrosinase mRNA 발현에서의 억제 효과를 확인하기 위하여 RT-PCR을 이용하였으며, $CHCI_3$ 층에서 분리해낸 A 분획에서 tyrosinase mRNA 발현을 억제시킴을 확인하였다.

Cytokeratin의 RT-PCR 및 면역조직화학적 분석을 이용한 구강편평세포암종의 임파절 미세전이 진단과 예후인자 효용성 평가 (DIAGNOSIS OF MICROMETASTASIS IN LYMPH NODE AND CLINICAL EVALUATION OF PROGNOSTIC FACTOR OF ORAL SCC USING RT-PCR AND IMMUNOHISTOCHEMISTRY FOR CYTOKERATIN)

  • 박성진;이원덕;임구영;강진한;명훈;이종호;김명진
    • Journal of the Korean Association of Oral and Maxillofacial Surgeons
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    • 제31권2호
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    • pp.105-115
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    • 2005
  • Purpose: The lymph node status assessed by conventional histological examination is the most important prognostic factor in patients undergoing surgery for oral squamous cell carcinoma. The presence of lymph node metastasis has a strong adverse impact on patient survival even after extended radical resection. Despite these findings, tumour recurrence is not rare after surgery, even when histological examination shows no lymph node metastasis. Recently, molecular-genetically and immunohistochemically demonstrated micrometastasis to the lymph nodes has been shown to have a significant adverse influence on survival in patients with squamous cell carcinoma and histologically negative nodes. The present study sought to determine the incidence and clarify the clinical significance of molecular-genetically and immunohistochemically demonstrated nodal micrometastases and to correlate these data with the stage of oral cancer. Methods: Lymph nodes systematically removed from 71 patients who underwent curative resection between 1998 and 2003 with head and neck squamous cell carcinoma were examined molecular-genetically to detect cytokeratin 5 mRNA with RT-PCR and immunohistochemically to detect cells that stained positively for cytokeratins with the monoclonal antibody cocktail AE1/AE3. The postoperative course and survival rates were compared among patients with and without micrometastases, after numerical classification of overt metastatic nodes. Results: micrometastases were detected in 43(60%) of 71 patients by RT-PCR and 26(36%) of 71 patients by immunohistochemistry. By RT-PCR analysis, patients exhibiting a positive band for CK 5 mRNA had a significantly worse prognosis than those were RT-PCR negative. By immunohistochemistry, the presence of micrometastasis did not predict patient outcome. Conclusion: Micrometastases detected by RT-PCR may be of clinical value in identifying patients who may be at high risk for recurrence and who are therefore likely to benefit from systemic adjuvant therapy.

Characterization of Pathogenesis and Plant Defence-related Genes Against Potato virus X infection empolying Potato X virus expresssin vector

  • Park, Mi-Ri;Kwon, Sun-Jung;Kim, Kook-Hyung
    • 한국식물병리학회:학술대회논문집
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    • 한국식물병리학회 2003년도 정기총회 및 추계학술발표회
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    • pp.74.1-74
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    • 2003
  • Differential display (DD) of mRNA is a technique in which mRNA species expressed by a cell population are reverse transcribed and then amplified by many separate polymerase chain reactions (PCR). Using DD-RT-PCR we obtained many genes that expressed differentially in healthy and PVX-infected Nicotiana benthamima, using total RNAs extracted from healthy and PVX-infected N. benthamiana plants. Three hundred and twenty-five DNA fragments isolated from DD-RT-PCR were cloned and sequenced for further characterization. Several host genes including SKPI-like protein, heat shock transcription factor and Avr9/Cf-9 rapidly elicited protein were selected to obtain full-length open reading frame and to characterize their potential involvement in virus disease development and/or host's defense against virus infection employing PVX-based expression vector. Transcrips from wild-type and clones containing each selected gene were inoculated onto N. benthamiana Levels of virus replication were confirmedby RT-PCR and RNA blot analysis, Expression profiles and potential role(s) of selected genes upon PVX infection will be discussed.

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대장암 세포주에서 genistein과 daidzein의 병합처리에 의한 상승적인 세포독성 효과 (Synergistic Cytotoxic Effects by Combination Treatment of Genistein and Daidzein in Human Colorectal Cancer Cell)

  • 손성민;임승현;김효림;정민정;김태완;이종화;김종식
    • 생명과학회지
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    • 제19권9호
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    • pp.1294-1298
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    • 2009
  • 콩의 대표적인 이소플라본인 genistein과 daidzein에 의해 암세포 생존율에 미치는 영향을 확인하기 위하여, HCT116 세포주에 genistein과 daidzein을 농도 의존적으로 처리하였다. Genistein은 처리한 농도 의존적으로 암세포 생존율을 감소시켰으며, 이에 반해 daidzein은 세포생존율에 큰 변화를 보여주지는 못하였다. 이전의 마이크로어레이 실험 결과에 의하면, $50{\mu}M$의 genistein에 의해 2배 이상 증가되는 유전자 71개, 2배 이상 감소되는 유전자 64개가 검색되었다. 이중 3개의 유전자(DKK-1, ATF3 그리 고 NAG-1)를 선택하여, 마이크로어레이 실험 결과를 검증하기 위하여 RT-PCR을 수행하였다. RT-PCR 결과 마이크로어레이 결과와 모두 일치함을 증명하였다. 한편, genistein과 daidzein에 의한 병합처리에 의해 암세포생존에 미치는 영향을 확인하였다. 그 결과 병합처리에 의한 상승적인 세포독성 효과를 확인하였다. RT-PCR과 real-time PCR의 결과 genistein과 daidzein의 병합처리에 의해 항암유전자인 NAG-1 유전자가 상승적으로 발현이 증가됨을 확인하였다. 이러한 결과는 이소플라본뿐만 아니라 대두제품에 의한 암 화학예방법의 기전을 이해하는 도움을 줄 것으로 생각된다.

국내 옥수수 순계주에서 CP4 5-Enol- Pyruvylshikimate-3- Phosphate Synthase 유전자의 발현 (Expression of CP4 5-Enol-Pyruvylshikimate-3- Phosphate Synthase Transgene in Inbred Line of Korean Domestic Maize (Zea may L.))

  • 조미애;권석윤;김진석;이병규;문추연;최필선
    • Journal of Plant Biotechnology
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    • 제34권4호
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    • pp.375-380
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    • 2007
  • 국내 옥수수 순계주에서 Agrobacterium 공동배양으로 CP4 5-Enol-pyruvylshikimate-3-phosphate synthase (CP4 EPSPS) 유전자가 도입된 제초제저항성식물체를 개발하였다. 5개의 순계주 (HW1, KL103, HW3, HW4, HW7)의 미숙배를 Ubiquitin promoter-CP4 EPSPS 유전자와 CaMV35S promoter-nptII 유전자가 발현되도록 제조된 pCAMBIA2300 벡터를 C58C1 Agrobacterium에 형질전환하여 공동 배양하였다. 항생제로 paromomycin이 첨가된 배지에서 선발된 옥수수 형질전환체를 PCR, RT-PCR 및 Northern 분석을 통하여 유전자의 도입과 발현을 확인하였다. 또한 형질전환 식물체의 glyphosate 처리에 따른 shikimate 축적반응을 확인하였다. Paromomycin 저항성 캘러스 형성빈도는 옥수수 각 순계주 HW1, KL103, HW3, HW4, HW7에서 각각 0.37%, 0.03%, 2.20%, 2.37%, 0.81%로 나타났으며, PCR분석을 통하여 최종적으로 2개의 옥수수 순계주 (HW3, HW4)의 paromomycin 저항성 캘러스로부터 분화된 식물체에서 확인하였다. 이러한 형질전환체중에서 RT-PCR 및 Nothern blot 분석을 통하여 CP4 EPSPS 유전자가 발현되는 2개의 계통 (M266, M104) 을 선발하였고, shikimate 축적반응을 통하여 glyphosate에 대한 저항성을 갖는 계통 (M266)을 최종적으로 선발하였다. 이러한 결과는 국내 옥수수 순계주에서 제초제저항성을 갖는 옥수수 형질 전환체를 개발할 수 있음을 시사한다.

Gene Expression of Heart and Adipocyte Fatty Acid-binding Protein in Chickens by FQ-RT-PCR

  • Tu, Yunjie;Su, Yijun;Wang, Kehua;Zhang, Xueyu;Tong, Haibing;Gao, Yushi
    • Asian-Australasian Journal of Animal Sciences
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    • 제23권8호
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    • pp.987-992
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    • 2010
  • This study was to detect the expression of heart fatty acid-binding protein (H-FABP) and adipocyte fatty acid-binding protein (A-FABP) gene mRNA in different tissues of Rugao and Luyuan chickens at 56 d and 120 d by real-time fluorescence quantitative reverse transcription polymerase-chain reaction (FQ-RT-PCR). The primers were designed according to the sequences of HFABP, A-FABP and GAPDH genes in Gallus gallus, which were used as target genes and internal reference gene, respectively. The levels of H-FABP and A-FABP gene expression were detected by SYBR Green I FQ-RT-PCR. The relative H-FABP and A-FABP gene mRNA expression level was calculated with 2-$^{{\Delta}Ct}$. Melting curve analysis showed a single peak of three genes. Intramuscular fat (IMF) content in breast muscle and leg muscle of the two chicken breeds at 120 d was higher than at 56 d. IMF content in breast muscle and leg muscle at 56 d and 120 d in Luyuan was significantly higher than in Rugao, however, abdominal fat of Luyuan was significantly lower than that of Rugao. The relative H-FABP gene mRNA expression level in cardiac muscle was the highest in both chicken breeds. The relative H-FABP and A-FABP gene expression of different tissues in Luyuan was higher than in Rugao. H-FABP gene mRNA expression had a negative effect on IMF of leg and breast muscles, and was significantly negatively correlated with IMF content. The relative A-FABP gene mRNA level in abdominal fat was higher than in liver. The A-FABP gene mRNA was not expressed in leg, breast and cardiac muscles. A-FABP gene mRNA expression level was significantly positively correlated with abdominal fat and had a significant effect on abdominal fat but not IMF content.

Comparison of Mycobacterium tuberculosis Specific Antigen Stimulation Time for Performing Interferon Gamma mRNA Assay for Detecting Latent Tuberculosis Infection

  • Kim, Sunghyun;Cho, Jang-Eun;Kim, Hyunjung;Lee, Dongsup;Jeon, Bo-Young;Lee, Hyejon;Cho, Sang-Nae;Kim, Young Keun;Lee, Hyeyoung
    • 대한의생명과학회지
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    • 제19권2호
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    • pp.90-97
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    • 2013
  • The tuberculin skin test (TST) and interferon gamma (IFN-${\gamma}$) release assay (IGRA) have been widely used for diagnosis of latent tuberculosis infection (LTBI). In order to overcome limitations of current LTBI diagnostic methods, the development of a novel molecular assay which is able to measure the IFN-${\gamma}$ messenger RNA (mRNA) expression level after stimulation with Mycobacterium tuberculosis (MTB) specific antigen was recently developed. The ability of a molecular assay to detect MTB infection was similar to commercial IGRA however, the optimal incubation time for stimulating IFN-${\gamma}$ was not yet established. Therefore, in this study the direct comparisons of MTB Ag stimulation times (4 and 24 hrs) were performed for diagnosis of MTB infection. Data showed that the coincident rate between QFT-GIT IFN-${\gamma}$ ELISA and IFN-${\gamma}$ RT-PCR (4 hrs) was 88.35% and that of QFT-GIT and IFN-${\gamma}$ RT-PCR (24 hrs) was 70.85%. Based on a receiver operating characteristic (ROC) curve, the 4 hrs-MTB specific Ag stimulation time for IFN-${\gamma}$ RT-PCR had the significant P value, 95% CI value, and AUC (P < 0.0001, 95% CI=0.82 to 1.02, and AUC=0.9214) in comparison with 24 hrs-MTB specific Ag stimulation time (P = 0.009, 95% CI=0.06 to 0.94, and AUC=0.7711). These results show that 4-hr was the most optimal MTB Ag stimulation time for performing IFN-${\gamma}$ RT-PCR. Although semi-quantitative RT-PCR had a few analytical limitations, it might be useful as an alternative molecular diagnostic method for detecting MTB infection.

치수 및 치근단 질환에서의 단백분해효소 및 단백분해효소 억제제의 활성도에 관한 연구 (A STUDY ON THE ACTIVITY OF PROTEINASE AND PROTEINASE INHIBITOR IN PULPAL AND PERIAPICAL PATHOSES)

  • 김진우;;임성삼
    • Restorative Dentistry and Endodontics
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    • 제25권4호
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    • pp.509-526
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    • 2000
  • It is known that injuries to the dentin have a corresponding inflammatory effect on the pulp and these inflammatory effects frequently result in pulpal pathoses due to progressive degradation of pulpal connective tissue. It was supposed that the tissue degradation in different inflammatory process was controlled by proteinase activity and antiproteinase activity. Therefore, the purpose of this study was to examine the pulp and periapical pathoses in terms of the activities of proteinase and proteinase inhibitor, 37 pulpal tissues were divided by clinical diagnostic criteria into normal pulp, acute inflamed pulp, and chronic inflamed pulp, and then those groups were subdivided by histopathological findings into 5 pulpal pathoses groups, i.e. normal pulp (P1, n=8), chronic pulpitis with fibrotic change (P2, n=2), chronic pulpitis with dystrophic calcification (P3, n=11), chronic pulpitis with pulp abscess (P4, n=7), acute pulpitis with necrotic change (P5, n=4), 26 periapical tissues were also divided by ordinary histopathological findings into 3 periapical pathoses group, i.e., granuloma (A1, n=17), cyst (A2, n=2) and abscess (A3, n=7). The activities of proteinases (cathepsin G, MMP-3) and proteinase inhibitors (${\alpha}1$-AT, TIMP-1 and, SLPI) were evaluated by RT-PCR and immunohistochemical methods. The results were as follows. 1. Generally, the intensity of immunohistochemical staining of proteinases and proteinase inhibitors increased in P2 and P5 groups compared to P1 group. 2. The immunohistochemical stain of proteinases and proteinase inhibitors was intensely detected in P2 group, showing low inflammatory reaction and low tissue degradation, but it was reduced in P3 and P4 groups, showing severe tissue degradation. 3. The distribution of proteinases and proteinase inhibitors in pulpal pathoses was consistently presented by immunohistochemical staining, while the expression of proteinase and/or proteinase inhibitors mRNAs in pulpal pathoses was occasionally detected by RT-PCR methods. 4. RT-PCR of proteinase and proteinase inhibitors was usually positive in P2, showing rare tissue degradation, but it was almost negative in P3 and P4, showing severe tissue degradation. 5. We presume that the reason why the level of proteinase and proteinase inhibitors was so sparse in RT-PCR method is due to the abrupt decrease of mRNA synthesis or degradation of synthesized mRNA of proteinase and/or proteinase inhibitors depend on the inflammatory reaction and/or on the degradation of pulp tissues(P3, P4). 6. Pulpal pathoses groups showed significant lower RT-PCR detection of proteinases and proteinase inhibitors than the periapical pathoses group(p<0.05), and there is no significant difference among the periapical pathoses groups(p>0.05).

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Development of Recombinant Coat Protein Antibody Based IC-RT-PCR and Comparison of its Sensitivity with Other Immunoassays for the Detection of Papaya Ringspot Virus Isolates from India

  • Sreenivasulu, M.;Gopal, D.V.R. Sai
    • The Plant Pathology Journal
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    • 제26권1호
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    • pp.25-31
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    • 2010
  • Papaya ringspot virus (PRSV) causes the most widespread and devastating disease in papaya. Isolates of PRSV originating from different geographical regions in south India were collected and maintained on natural host papaya. The entire coat protein (CP) gene of Papaya ringspot virus-P biotype (PRSV-P) was amplified by RTPCR. The amplicon was inserted into pGEM-T vector, sequenced and sub cloned into a bacterial expression vector pRSET-A using a directional cloning strategy. The PRSV coat protein was over-expressed as a fusion protein in Escherichia coli. SDS-PAGE gel revealed that CP expressed as a ~40 kDa protein. The recombinant coat protein (rCP) fused with 6x His-tag was purified from E.coli using Ni-NTA resin. The antigenicity of the fusion protein was determined by western blot analysis using antibodies raised against purified PRSV. The purified rCP was used as an antigen to produce high titer PRSV specific polyclonal antiserum. The resulting antiserum was used to develop an immunocapture reverse transcription-polymerase chain reaction (IC-RT-PCR) assay and compared its sensitivity levels with ELISA based assays for detection of PRSV isolates. IC-RT-PCR was shown to be the most sensitive test followed by dot-blot immunobinding assay (DBIA) and plate trapped ELISA.

The Expression Patterns of Estrogen-responsive Genes by Bisphenol A in the Wild Medaka (Oryzias sinensis)

  • Lee, Chul-Woo;Park, Min-Kyung;Kim, Hyun-Mi;Kim, Hak-Joo;Choi, Kyung-Hee
    • Molecular & Cellular Toxicology
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    • 제3권3호
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    • pp.185-189
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    • 2007
  • Gene expression levels of choriogenin, vitellogenin and estrogen receptor were determined using Reverse transcription (RT)-PCR technique after exposure to estrogenic chemical bisphenol A in the Korean wild medaka (Oryzias sinensis). These genes have been known to be induced in male test fish when the fish are exposed to estrogenic chemicals. Therefore they can be suggested as a possible biomarker of endocrine disruption in fish, however, relatively little has been known about these genes expression by estrogenic chemicals in Korean wild fish. Mature male Oryzias sinensis were treated with bisphenol A at nominal concentrations of 0.02, 0.2 and 2 mg/L for 6 days and total RNA was extracted from the livers of treated fish for RT-PCR. When the five biomarker genes were amplified by RT-PCR in the same condition, mRNA induction level of each gene was elevated with different sensitivities. Conclusively, the results of this work indicated that measurement of vitellogenin and choriogenin using RT-PCR is effective as a simple tool for the screening of estrogenic chemicals and suggested that O. sinensis would be a suitable model fish for the environmental risk assessment of potential endocrine disruptors.