• Title/Summary/Keyword: Competitive RT-PCR

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Zinc Status Assessment by Analysis of Mononuclear Cell Metallothionein mRNA Using Competitive-Reverse Transcriptase-Polymerase Chain Reaction

  • Lee, Soo-Lim;Yoon, Jin-Sook;Kwon, Chong-Suk;Beattie, John H.;Kwun, In-Sook
    • Preventive Nutrition and Food Science
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    • v.9 no.3
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    • pp.276-282
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    • 2004

  • Marginal Zn deficiency is prevalent through the world and yet human zinc status has not been properly assessed due to the lack of a reliable diagnostic indicator. One potential possibility for zinc status assessment using Zn-binding protein, metallothionein (MT)-mRNA, has been proposed. The purpose of the present study was aimed to show whether measurement of mononuclear cell (MNC) MT mRNA, using a competitive-reverse transcriptase-polymerase chain reaction (competitive-RT-PCR) assay, could indicate zinc status in human subjects. In this study, MNC MT-mRNA expression was measured using a competitive-RT-PCR to compare before and after 14 days of zinc supplementation (50 mg Zn/das zinc gluconate). RT-PCR oligonucleotide primers which were designed to amplify both a 278 bp segment of the human MT-2A cDNA and a 198 bp mutant competitor cDNA template from MNCs, were prepared. MT-2A mRNA was normalized by reference to the housekeeping gene, $\beta$-actin, mRNA for which was also measured by competitive-RT-PCR. There was considerable inter-individual variation in MT-mRNA concentration and yet, the mean MT-2A mRNA level increased 4.7-fold after Zn supplementation, as compared to before Zn supplementation. This MT-2A mRNA level was shown as the same pattern and, even more sensitive assay, compared to the conventional plasma and red blood cells (RBCs) Zn assessment in which plasma and RBCs zinc levels increased 2.3- and 1.2-fold, respectively (p<0.05). We suggest that MT competitive-RT-PCR can be a useful assessment tool for evaluating human zinc status.

  • https://doi.org/10.3746/jfn.2004.9.3.276 인용 PDF KSCI

Changes of splenocyte $IFN-{\gamma}$ mRNA synthesis in rats infected with Paragonimus westermani

  • Cho, Jun-Kyong;KWon, Hye-Soo;Joo, Kyoung-Hwan;Lee, Joon-Sang;Cho, Sung-Weon
    • Parasites, Hosts and Diseases
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    • v.37 no.4
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    • pp.285-287
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    • 1999

  • Changes in the expression level of splenocyte $IFN-{\gamma}$mRNA of Sprague-Dawley (SD) rats infected with Paragonimus westermani were analyzed by competitive reverse transcription-polymerase chain reaction (RT-PCR) followed by southern blot. The template RNA was extracted from the splenocytes of rats infected with 20 metacercariae of P. westermani. The products of competitive RT-PCR were subjected to southern blot and enhanced chemiluminescence (ECL), and analyzed with a densitometer. In comparison with that of uninfected control rat splenocytes (value of 1), the levels of mRNA expression of $IFN-{\gamma}$had changed to 0.747 at 1 week post infection (PI), 0.00175 at 2 week PI, 0.0217 at 3 week PI, 0.194 at 4 week PI and then to 0.537 at 5 week PI. The level at 7 week PI had returned to 1.25, comparable with that of uninfected rats. These results show that, when infected with p. westermani, the levels of $IFN-{\gamma}$ mRNA of SD rat splenocytes were remarkably reduced by more than 500 times at 2 week PI and restored to normal level at 7 week PI.

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Repression of HspA2 mRNA Expression in Human Testes with Abnormal Spermatogenesis (비정상적 정자형성 환자의 정소에서 Heat Shock Protein A2 (hspA2) mRNA 발현의 감소)

  • Son, W.Y.;Hwang, S.H.;Han, C.T.;Lee, J.H.;Kim, S.J.;Kim, Y.C.
    • Clinical and Experimental Reproductive Medicine
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    • v.26 no.1
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    • pp.103-109
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    • 1999

  • Objective: Heat shock protein 70-2 (Hsp70-2) gene knockout mice are found to have premeiotic arrest at the primary spermatocyte stage with a complete absence of spermatids and spermatozoa. This observation led to the hypothesis that hspA2 may be disrupted in human testes with abnormal spermatogenesis. To test this hypothesis, we studied the mRNA expression of hspA2 in infertile men with azoospermia. Design: The mRNA expression were analyzed by competitive RT-PCR among testes with normal spermatogenesis, pachytene spermatocyte arrest, and sertoli-cell only syndrome. Materials and methods: Testicular biopsy was performed in men with azoospermia (n=15). Specimens were subdivided into three groups: (group 1) normal spermatogenesis (n=5), (group 2) spermatocyte arrest (n=5), (group 3) Sertoli-cell only syndrome (n=5). Total RNA was extracted by Trizol reagent. Total extracted RNA was reverse transcribed into cDNA and amplified by PCR using specific primers for hspA2 target cDNAs. A competitive cDNA fragment was constructed by deleting a defined fragment from the target cDNA sequence, and then coamplified with the target cDNA for competitive PCR. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as an internal control. Results: On Competitive RT-PCR analyses for hspA2 mRNA, significant amount of hspA2 expression was observed in group 1, whereas a constitutively low level of hspA2 was expressed in groups 2 and 3. Conclusion(s): The study demonstrates that the hspA2 gene expression is down-regulated in human testes with abnormal spermatogenesis, which in turn suggests that hspA2 gene may play a specific role during meiosis in human testes.

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Expression of rpr, grim, dcp-1, diapl, and diap2 during Drosophila Development (노랑초파리 발생과정에서 rpr, grim, dcp-1, diapl, diap2의 발현)

  • Park, Ji-Gweon;Chung, Ki-Wha;Kim, Se-Jae
    • Development and Reproduction
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    • v.5 no.2
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    • pp.131-136
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    • 2001

  • The developmental profiles of rpr, grim, dcp-1, diapl, diap2 transcripts, which were involved in programmed cell death, were analyzed using competitive RT-PCR in whole animals during Drosophila development. The fluctuation patterns of transcript levels of the apoptotic initiators(rpr and grim) were similar to those of the ecdysone titer in Drosophila life cycle. The transcript of dcp-1, which is considered as effector caspase, was expressed strong1y at early embryo and female adult stages. However, the transcript levels of anti-apoptotic factors diap1 and diap2, showed the reverse pattern comparing with those of apoptotic factors(rpr and grim). Also, the transcript levels of rpr, diap2 and dcp-1 were quantified in the salivary glands and wing discs dissected from the wandering late third instar larva. The transcript levels of rpr and diap2 were changed reversely each other in both tissues from wandering stage to puparium formation. These results suggest that the expressions of cell death related genes are regulated by the ecdysone signals during normal development.

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