• 한국가축번식학회지
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• 제18권3호
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• pp.199-206
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• 1994

본 연구는 생쥐 배 발생과정의 상이한 발현을 RT-PCR법에 의해 무작위 증폭함으로서 새로운 유전자를 손쉽게 크로닝하기 위해 수행되었다. mRNA의 상이한 display법은 Ling 과 Pardee (Science 257, 1992)에 의해 개발되었으며, 최근 Zimermann과 Schultz (PNAS USA 91, 1994)에 의해 재증명되었다. 이 방법은 특정 유전자의 일시적 발현의 변화가 maternal 제어로부터 접합체 제어로의 이행에 따른 발현전이, 다정자 침입과 단일 정자 침입에 의한 배발생의 기능적 차이, 성공적으로 부화한 배반포기 배와 부화에 실패한 배반포기 배에서의 발현의 차이는 물론 세포주기에 따른 유전자 발현 양식의 변화에 따른 새로운 유전자의 크로닝을 가능케 한다. 이 방법에 의해, 2세포기 특이 발현 유전자를 크로닝 하였으며, 이 유전자는 EcoRI제한 효소 처리후 Southern blot을 행한 결과 약 15kb genomic size를 가진 것으로 나타났다. 이 새로운 유전자는 간장 특이적 발현을 나타내었다. 또한, 적어도 2개의 mRNA가 존재하였으며, 이는 RNA splicing에 의한 것으로 추정되었다. (PCR, RT-PCR, cloning, preimplantation, mouse)

• Clinical and Experimental Reproductive Medicine
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• 제28권3호
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• pp.183-190
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• 2001

Objective: Recently, microdissection of tissue sections has been used increasingly for the isolation of morphologically identified homogeneous cell populations, thus overcoming the obstacle of tissue complexity for the analysis cell-specific expression of macromolecules. The aim of the present study was to establish the minimal conditions required for the RNA extraction and amplification from the cells captured by the laser captured microdissection. Methods : Mouse ovaries were fixed and cut into serial sections (7 im thickness). Oocytes were captured by laser captured microdissection (LCM) method by using PixCell $II^{TM}$ system. The frozen sections were fixed in 70% ethanol and stained with hematoxylin and eosin, while the paraffin sections were stained with Multiple stain. Sections were dehydrated in graded alcohols followed by xylene and air-dried for 20 min prior to LCM. All reactions were performed in ribonuclease free solutions to prevent RNA degradation. After LCM, total RNA extraction from the captured oocytes was performed using the guanidinium isothiocyanate (GITC) solution, and subsequently evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR) for glyceraldehyde-3-phosphate-dehydrogenase (GAPDH). Results: With the frozen sections, detection of the GAPDH mRNA expression in the number of captured 25 oocytes were not repeatable, but the expression was always detectable from 50 oocytes. With 25 oocytes, at least 27 PCR cycles were required, whereas with 50 oocytes, 21 cycles were enough to detect GA PDH expression. Amount of the primary cDNA required for RT-PCR was reduced down to at least 0.25 $\grave{i}$ l with 50 oocytes, thus the resting 19.75 il cDNA can be used for the testing other interested gene expression. Tissue-to-slide, tissue-to-tissue forces were very high in the paraffin sections, thus the greater number of cell procurement was required than the frozen sections. Conclusion: We have described a method for analyzing gene expression at the RNA level with the homogeneously microdissected cells from the small amount of tissues with complexity. We found that LCM coupled with RT-PCR could detect housekeeping gene expression in 50 oocytes captured. This technique can be easily applied for the study of gene expression with the small amount of tissues.

• Journal of Gastric Cancer
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• 제3권3호
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• pp.128-133
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• 2003

Purpose: The benefits of the 'no-touch' isolation techniquethat is usually performed to prevent the circulation of tumor cells are not evident. The aim of this study was to determine whether the no-touch isolation technique for treating gastrointestinal cancers could prevent the circulation of tumor cells detected by reverse transcriptase polymerase chain reaction (RT-PCR). Matrials and Methods: By using RT-PCR to amplify mRNAs for two specific epithelial markers, carcinoembryonic antigen (CEA) and cytokeratin 20 (CK-20), we examined 34 gastric cancer patients who had been histologically diagnosed and 22 patients had undergone serosal and peritoneal brushing. Results: In 10 ($29.4\%$) of the 34 gastric cancer patients, we detected CK20 mRNA before manipulation, and in 17 ($51.5\%$) of those patients, after we detected it. The density of the CK20 mRNA band was increased in 11 cases ($33.3\%$) and the density was decreased in 2 cases ($6.1\%$). In 16 ($48.5\%$) of the 34 gastric cancer patients, we detected CEA mRNA before manipulation, and in 17 ($51.5\%$) patients after we detected it. The density of the CEA mRNA band was increased in 8 cases ($24.2\%$) and decreased in 3 cases ($9.1\%$). Conclusion: These result suggest that the ' no-touch isolation technique ' might be useful when operating on advanced gastric cancer patients and that serosal or Douglas pouch brushing can be used to determine the status of micrometastasis.

• 대한의생명과학회지
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• 제6권2호
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• pp.141-149
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• 2000

우리 나라에서 발생하고 있는 급성 출혈성 질환인 신증후출혈열의 원인 바이러스는 Family Bunyaviridae의 Genus Hantavirus에 속하는 한탄과 서울바이러스에 의하여 발생되고 있다. 본 연구에서는 신증후출혈열로 의뢰된 환자에서 한탄바이러스에 대한 항체가를 간접면역형광항체법(indirect immunofluorescent antibody technique, IFAT), 면역효소측정법 (enzyme-linked immunosorbent assay, ELISA) (IgG, IgM), 고비중입자응집반응 (high density composite particle agglutination, HDPA) 및 플라크감소중화시험 (plaque reduction neutralization test, PRNT) 등으로 비교 측정하였고, 신증후출혈열 환자로 확진된 15명의 한탄바이러스 혈청형을 PRNT와 혈청형 특이 역전사 효소 중합효소연쇄반응(nested reverse transcriptase polymerase chain reaction, nested RT-PCR)으로 확인하였다. 신증후출혈열로 의뢰된 환자에서의 한탄바이러스에 대한 IFAT, ELISA (IgG, IgM), HDPA그리고 PRNT비교에서 형광항체, ELISA IgG,응집항체 및 중화항체는 8명 모두 높게 나타났으며, ELISA IgM은 5명에서는 현저히 높은 항체를 보유하고 있었다. 신증후출혈 열 환자 15명에서는 높은 형광항체와 중화항체 역가를 나타내었고, 15명 중 12명은 한탄바이러스, 2명은 서울바이러스에 대한 높은 중화항체를 갖고 있었으며, 1명은 두 바이러스에 대하여 동일한 항체 역가를 나타내었으며, 혈청형 특이 primer를 사용한 nested RT-PCR에서는 15명 중 3명과 1명만이 한탄바이러스와 서울바이러스 primer에 대해 RNA가 검출되었다.

• 한국가축번식학회지
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• 제19권1호
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• pp.55-63
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• 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.

• 한국자원식물학회지
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• 제16권1호
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• pp.40-48
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• 2003

C. destructans는 인삼에서 가장 문제가 되고 있는 뿌리섞음병을 유발하는 매우 중요한 미생물이다. 현재까지 정상적인 인삼포장이나 폐포지에서도 이 병원균의 농도를 조사할 만한 방법이 없어 이를 쉽게 조사함으로서 인삼 예정지 관린시 도움을 줄 수 있는 새로운 방법이 절실이 요구되고 있다. 본 연구에서는 nested PCR이란 분자생물학적 방법을 이용하여 효과적으로 매우 낮은 농도의 C. destructans을 검출할 수 있는 방법을 개발하였다. 2개의 universal ITS primers(ITS5F와 ITS4R)을 사 용 하 여 Cylindrocarpon spp.의 rDNA로부터 ITS영역을 증폭하였다. 이어 C. destructans의 specific primer(Nest 1 과 Nest 2)을 사용하여 최적의 PCR조건으로 재증폭시켜 밴드를 확인하였다. 또한 이런 2번의 과정을 4개의 primer를 동시에 사용함으로서 한번에 확인할 수 있는 방법을 개발하였으며 이에 따른 PCR조건도 확립하였다. 따라서 본 방법에 의해서 인삼포장의 토양에서 채취된 매우 낮은 농도의 wild type C. destructans spore로부터 성공적으로 positive band을 확인함으로써 추후 인삼포장의 선정 및 4년생에서 6년까지(홍삼포) 재배기간등의 예측에 활용 될 것으로 생각된다.

• The Plant Pathology Journal
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• 제26권3호
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• pp.286-288
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• 2010

Plant tissue homogenization using a mortar or mechanical equipment has been the preferred method for obtaining high yields of total RNA; this method, however, is both time-consuming and expensive. Additionally, homogenization may generate excessive endogenous RNases, polyphenolics, and other substances that reduce the quality and quantity of RNA. In this study, we describe the microwave irradiation-assisted RNA extraction (MIRE) technique which, without tissue disruption and homogenization, allows for the cost-effective and rapid generation of intact RNA from apple cane shavings and the reliable detection of apple virus by RT-PCR.

• Toxicological Research
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• 제13권4호
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• pp.417-421
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• 1997

Synaptobrevin is a kind of vesicle associated membrane proteins (VAMPs) which plays a secretary role in the neuronal synapse and was recently known as the biochemical target of botulinum neurotoxin type B. The structural gene of the synaptobrevin was cloned from mouse brain using RT-PCR technique and was seqrtenced. The deduced amino acid sequence showed that the synaptobrevin protein from mouse brain is exactly the same with that of the rat brain in the amino acid level. The synaptobrevin gene was subcloned into pET3a vector and expressed in E. coli. The molecular weight of the recombinant protein was 19 kDa as expected. Moreover, when the recombinant synaptobrevin protein was incubated with the native neurotoxin of Clostridium botulinum type B, it was cleaved by the toxin in a time dependent manner. This implies that the recombinant synaptobrevin protein and the native toxin are reacted in the same way as the native synaptobrevin did in the neuronal cells.

• 한국해양바이오학회지
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• 제2권2호
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• pp.77-80
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• 2007

Macroalgal tissues can be used as indicating materials for environmental assessment using several algal biotechnology techniques. As bioassay test organisms, macroalgal tissues are required as an axenic state for suitable biological indicators. Callus formation and blade regeneration under suitable culture conditions are also useful for the tests. Quantitative method using tetrazolium chloride or $alamarBlue^{TM}$ is devised on a rapid assessment of the seaweed viability. The use of RT-PCR especially differential display technique should provide the means for the detection and isolation of the responding genes induced by the environmental stress. Seaweed thriving in more environmental changes might contain more diverse biologically active substances.

• Journal of Animal Science and Technology
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• 제46권3호
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• pp.315-324
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• 2004

본 연구는 돼지에서 특이적으로 만들어지는 것으로 알려진 19-nortestosterone(nandrolone) 및 여성호르몬(estrogen)의 합성에 관여하는 효소인 Cytochrome P450 aromatase에 대한 유전자의 발현 양상을 밝혀내기 위해 실시되었다. RT-PCR과 최근에 개발된 DHPLC(Denature High Performance Liquid Chromatography 또는 WAVE라고 함) 분석 장치를 이용하여 정소와 난소에서 어떤 isoform의 aromatase 유전자가 발현되는지에 관해 조사하였다. 이러한 방법을 통해 같은 양의 RNA 중에 존재하는 정소내 aromatase mRNA가 난소보다 상대적으로 많이 존재한다는 것이 밝혀졌으며, 이는 돼지의 경우 수컷이 암컷 보다 혈중 여성호르몬이 더 높게 나타난다는 이전의 연구 발표가 돼지에서 여성호르몬을 만드는 aromatase 유전자가 난소에 비해 정소에서 더 많이 만들어지기 때문이라는 사실을 입증하였다. 또한, 정소와 난소에서 발현되는 aromatse 유전자를 PCR를 이용하여 증폭한 후 DHPLC를 이용하여 분석한 결과 type II, III와 다르다는 것을 확인하였다. RT-PCR에 의해 증폭된 aromatase DNA 단편을 plasmid vector에 cloning한 후에 그 염기 서열을 분석한 결과, 정소 및 난소에서 발현되는 aromatse는 모두 type I(난소형)으로 밝혀졌다. 이는 어떻게 정소와 난소의 두 다른 조직에서 같은 aromatase 효소로부터 다른 steroid가 만들어 질 수 있는지에 대한 새로운 의문을 제시하는 연구결과이며, 현재 추가적인 연구가 진행 중이다. 또한, DHPLC 기술을 활용하여 염기서열이 매우 유사한 isoform 유전자들의 발현을 관찰할 수 있다는 사실이 증명되었다.