• 한국축산식품학회지
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• 제35권3호
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• pp.382-388
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• 2015

A duplex real-time polymerase chain reaction (PCR) based assay for the detection of porcine and horse meat in sausages was designed by using EvaGreen fluorescent dye. Primers were selected from mitochondrial 12S rRNA and 16S rRNA genes which are powerful regions for identification of horse and porcine meat. DNA from reference samples and industrial products was successfully extracted using the GIDAGEN® Multi-Fast DNA Isolation Kit. Genomes were identified based on their specific melting peaks (Mp) which are 82.5℃ and 78℃ for horse and porcine, respectively. The assay used in this study allowed the detection of as little as 0.0001% level of horse meat and 0.001% level of porcine meat in the experimental admixtures. These findings indicate that EvaGreen based duplex realtime PCR is a potentially sensitive, reliable, rapid and accurate assay for the detection of meat species adulterated with porcine and horse meats.

• Biotechnology and Bioprocess Engineering:BBE
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• 제9권1호
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• pp.23-34
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• 2004

Escherichia coli transcription termination factor Rho catalyzes the unwinding of RNA/DNA duplex in reactions that are coupled to ATP binding and hydrolysis. Fluorescence stopped-flow methods using ATP and the fluorescent 2'(3')-O-( N-methylanthraniloyl) derivatives (mant-derivatives) of ATP and ADP were used to probe the kinetics of nucleotide binding to and dissociation from the Rho-RNA complex. Presteady state nucleotide binding kinetics provides evidence for the presence of negative cooperativity in nucleotide binding among the multiple nucleotide binding sites on Rho hexamer. The binding of the first nucleotide to the Rho-RNA complex occurs at a bimolecular rate of 3.6${\times}$10$\^$6/ M$\^$-1/ sec$\^$-1/ whereas the second nucleotide binds at a slower rate of 4.7${\times}$10$\^$5/ M$\^$-1/ sec$\^$-1/ at 18$^{\circ}C$, RNA complexed with Rho affects the kinetics of nucleotide interaction with the active sites through conformational changes to the Rho hexamer, allowing the incoming nucleotide to be more accessible to the sites. Adenine nucleotide binding and dissociation is more favorable when RNA is bound to Rho, whereas ATP binding and dissociation step in the absence of RNA occurs significantly slower, at a rate ∼70- and ∼40-fold slower than those observed with the Rho-RNA complex, respectively.

• Asian-Australasian Journal of Animal Sciences
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• 제24권11호
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• pp.1529-1540
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• 2011

Silencing of a specific gene using RNAi (RNA interference) is a valuable tool for functional analysis of a target gene. However, information on RNAi for analysis of gene function in farm animals is relatively nil. In the present study, we have validated the interfering effects of siRNA (small interfering RNA) using both quantitative and qualitative gene silencing in buffalo granulosa cells. Qualitative gene knockdown was validated using a fluorescent vector, enhanced green fluorescence protein (EGFP) and fluorescently labeled siRNA (Cy3) duplex. While quantitatively, siRNA targeted against the luciferase and CYP19 mRNA was used to validate the technique. CYP19 gene, a candidate fertility gene, was selected as a model to demonstrate the technique optimization. However, to sustain the expression of CYP19 gene in culture conditions using serum is difficult because granulosa cells have the tendency to luteinize in presence of serum. Therefore, serum free culture conditions were optimized for transfection and were found to be more suitable for the maintenance of CYP19 gene transcripts in comparison to culture conditions with serum. Decline in fluorescence intensity of green fluorescent protein (EGFP) was observed following co-transfection with plasmid generating siRNA targeted against EGFP gene. Quantitative decrease in luminescence was seen when co-transfected with siRNA against the luciferase gene. A significant suppressive effect on the mRNA levels of CYP19 gene at 100 nM siRNA concentration was observed. Also, measurement of estradiol levels using ELISA (enzyme-linked immunosorbent assay) showed a significant decline in comparison to control. In conclusion, the present study validated gene silencing using RNAi in cultured buffalo granulosa cells which can be used as an effective tool for functional analysis of target genes.

• 한국미생물학회:학술대회논문집
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• 한국미생물학회 1998년도 제38회 춘계학술발표대회
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• pp.52.2-52.2
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• 1998

• 한국동물학회:학술대회논문집
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• 한국동물학회 2000년도 한국생물과학협회 학술발표대회
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• pp.90.2-91
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• 2000

No Abstract, See Full Text

• Genomics & Informatics
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• 제13권2호
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• pp.45-52
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• 2015

Acute myeloid leukemia is a well characterized blood cancer in which the unnatural growth of immature white blood cell takes place, where several genes transcription is regulated by the micro RNAs (miRNAs). Argonaute (AGO) protein is a protein family that binds to the miRNAs and mRNA complex where a strong binding affinity is crucial for its RNA silencing function. By understanding pattern recognition between the miRNAs-mRNA complex and its binding affinity with AGO protein, one can decipher the regulation of a particular gene and develop suitable siRNA for the same in disease condition. In the current work, HOXA9 gene has been selected from literature, whose deregulation is well-established in acute myeloid leukemia. Four miRNAs (mir-145, mir-126, let-7a, and mir-196b) have been selected to target mRNA of HOXA9 (NCBI accession No. NM_152739.3). The binding interaction between mRNAs and mRNA of HOXA9 gene was studied computationally. From result, it was observed mir-145 has highest affinity for HOXA9 gene. Furthermore, the interaction between miRNAs-mRNA duplex of all chosen miRNAs are docked with AGO protein (PDB ID: 3F73, chain A) to study their interaction at molecular level through an in silico approach. The residual interaction and hydrogen bonding are inspected in Discovery Studio 3.5 suites. The current investigation throws light on understanding of AGO-assisted miRNA based gene silencing mechanism in HOXA9 gene associated in acute myeloid leukemia computationally.

• Genomics & Informatics
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• 제14권3호
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• pp.112-124
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• 2016

Solid tumor is generally observed in tissues of epithelial or endothelial cells of lung, breast, prostate, pancreases, colorectal, stomach, and bladder, where several genes transcription is regulated by the microRNAs (miRNAs). Argonaute (AGO) protein is a family of protein which assists in miRNAs to bind with mRNAs of the target genes. Hence, study of the binding mechanism between AGO protein and miRNAs, and also with miRNAs-mRNAs duplex is crucial for understanding the RNA silencing mechanism. In the current work, 64 genes and 23 miRNAs have been selected from literatures, whose deregulation is well established in seven types of solid cancer like lung, breast, prostate, pancreases, colorectal, stomach, and bladder cancer. In silico study reveals, miRNAs namely, miR-106a, miR-21, and miR-29b-2 have a strong binding affinity towards PTEN, TGFBR2, and VEGFA genes, respectively, suggested as important factors in RNA silencing mechanism. Furthermore, interaction between AGO protein (PDB ID-3F73, chain A) with selected miRNAs and with miRNAs-mRNAs duplex were studied computationally to understand their binding at molecular level. The residual interaction and hydrogen bonding are inspected in Discovery Studio 3.5 suites. The current investigation throws light on understanding miRNAs based gene silencing mechanism in solid cancer.

• The Plant Pathology Journal
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• 제27권4호
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• pp.315-323
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• 2011

Soybean plants infected with Bean pod mottle virus (BPMV) develop acute symptoms that usually decrease in severity over time. In other plant-virus interactions, this type of symptom recovery has been associated with degradation of viral RNAs by RNA silencing, which is accompanied by the accumulation of virus-derived small interfering RNAs (siRNAs). In this study, changes in the accumulation of BPMV siRNAs were investigated in soybean plants infected with BPMV alone, or infected with both BPMV and Soybean mosaic virus (SMV) and in transgenic soybean plants expressing SMV helper component-protease (HC-Pro). In many potyviruses, HC-Pro is a potent suppressor of RNA silencing. In plants infected with BPMV alone, accumulation of siRNAs was positively correlated with symptom severity and accumulation of BPMV genomic RNAs. Plants infected with both BPMV and SMV and BPMV-infected transgenic soybean plants expressing SMV HC-Pro exhibited severe symptoms characteristic of BPMVSMV synergism, and showed enhanced accumulation of BPMV RNAs and siRNAs compared to plants infected with BPMV alone and nontransgenic plants. Likewise, SMV HC-Pro enhanced the accumulation of siRNAs produced from a silenced green fluorescent protein gene in transient expression assays, while the P19 silencing suppressor of Tomato bushy stunt virus did not. Consistent with the modes of action of HC-Pro in other systems, which have shown that HC-Pro suppresses RNA silencing by preventing the unwinding of duplex siRNAs and inhibiting siRNA methylation, these studies showed that SMV HC-Pro interfered with the activities of RNA-induced silencing complexes, but not the activities of Dicer-like enzymes in antiviral defenses.

• 생명과학회지
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• 제29권6호
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• pp.653-661
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• 2019

본 연구는 siRNA 기반 치료제등의 핵산치료제 개발에 있어서 필수적인 약물의 생체내 흡수, 분포, 대사, 배설에 대한 동태의 확인을 위해 stem-loop RT-qPCR 법을 이용하여 보다 더 정확한 시험법을 확립하고자 하였다. siRNA에 특이적인 primer와 probe를 선별하여 siRNA 정량검출 시험법을 최적화하였다. siRNA 표준시료를 이용하여 최적화된 시험법을 적용하였을 때 siRNA 표준시료에 대한 Cp 값(y)간의 선형분석 결과, 기울기 평균 -3.3, 결정계수 $R^2$>0.99으로 확인되어 siRNA 표준시료와 Cp 값 간의 회귀성이 매우 높아 정량 분석이 가능한 시험법임을 확인하였고, 같은 표준시료를 이용한 stem-loop RT-qPCR의 검출한계(LOD)는 10 fM, 최소정량한계(LLOQ)는 100 fM이었다. 확립된 시험법의 신뢰성을 확인하기 위해 시험자를 다르게 하고, 시험법을 3회 반복하여 각각 진행한 결과, siRNA 표준시료에 대한 Cp 값(y)간의 선형분석 결과 기울기와 결정계수 $R^2$의 재현성(slope ${\pm}-3.2$, 결정계수 $R^2$>0.99)을 확인하였고, 표준 곡선으로부터 환산된 siRNA 표준시료의 회수율(recovery ${\pm}20%$)과 완건성이 우수함을 확인하였다. 확립된 stem-loop RT-qPCR을 생체내 존재하는 약물 검증에 적용할 수 있는지 확인하기 위하여 시험동물에 siRNA를 주입 후 시간별 혈액을 채취하여 확립된 시험법으로 시험을 진행하였고 약물 동태학적 분석을 통해 siRNA치료제의 혈액내의 안정성을 확인하였다. 따라서 본연구에서 개발된 stem-loop RT-qPCR 분석법은 정확성, 정밀성 및 민감도가 높은 분석법으로 핵산치료제 개발 연구의 다양한 생체시료 분석 연구에 적용할 수 있을 것으로 기대한다.

• 한국인터넷방송통신학회논문지
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• 제16권6호
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• pp.135-140
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• 2016

본 논문에서는 유전정보 DNA 표준 코드인 [UC;AG]를 64괘로 분석했다. 인간의 유전정보를 담고 있는 DNA는 인산과 당에 A(아데닌) C(시토신) G(구아닌) T(티민) 등 네 종류의 염기가 30억쌍 이어 붙여진 형태이다. DNA 표준 코드를 64괘로 나타내고, 이 코드를 Kronecker product를 이용하여 $16{\times}4$행렬로 나타냈다. 이 $16{\times}4$ 행렬은 이중나선의 중복성을 가지고 있으며, 이 중복성을 제거하면 RNA코드 $4{\times}4$ 행렬을 얻는다. $16{\times}4$행렬은 Kronecker product를 이용하여 소 행렬로 분해되었다. DNA 이중나선을 행렬로 표시하고 유전정보 괘 배열 코드를 분석하였으며 그 예를 예 5, 6에 나타냈다.