• BMB Reports
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• 제40권2호
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• pp.226-231
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• 2007

Housekeeping genes are widely used as internal controls in a variety of study types, including real time RT-PCR, microarrays, Northern analysis and RNase protection assays. However, even commonly used housekeeping genes may vary in stability depending on the cell type or disease being studied. Thus, it is necessary to identify additional housekeeping-type genes that show sample-independent stability. Here, we used statistical analysis to examine a large human microarray database, seeking genes that were stably expressed in various tissues, disease states and cell lines. We further selected genes that were expressed at different levels, because reference and target genes should be present in similar copy numbers to achieve reliable quantitative results. Real time RT-PCR amplification of three newly identified reference genes, CGI-119, CTBP1 and GOLGAl, alongside three well-known housekeeping genes, B2M, GAPD, and TUBB, confirmed that the newly identified genes were more stably expressed in individual samples with similar ranges. These results collectively suggest that statistical analysis of microarray data can be used to identify new candidate housekeeping genes showing consistent expression across tissues and diseases. Our analysis identified three novel candidate housekeeping genes (CGI-119, GOLGA1, and CTBP1) that could prove useful for normalization across a variety of RNA-based techniques.

• 한국작물학회지
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• 제55권4호
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• pp.371-378
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• 2010

For sensitive and accurate gene expression analysis, normalization of gene expression data against housekeeping genes is required. There are conventional housekeeping gene (e.g. ACT) that primarily function as an internal control of transcription. In this study, we performed an in silico analysis of 278 rice gene expression samples (GSM) in order to identify the gene that is most consistently expressed. Based on this analysis, we identified novel candidate housekeeping genes that displayed improved stability among the cross experimental conditions. Furthermore four of the most conventional housekeeping genes were included in our 30 other housekeeping genes among the most stable genes. Therefore, these 30 genes can he used to normalize transcription results in gene expression studies on rice at a broad range of experimental conditions.

• 한국식품저장유통학회지
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• 제14권6호
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• pp.688-694
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• 2007

To evaluate the human risk of long-term intake of genetically modified (GM) rice, we carried out RT-PCR of housekeeping genes. Housekeeping genes, which show highly uniform expression in living organisms during various stages of development and under different environmental conditions, were normalized by RT-PCR. We assessed the expression of 10 common housekeeping genes (18s rRNA, 25S rRNA, UBC, UBQ5, UBQ10, ACT11, GAPDH, eEF-$1{\alpha}$, ${\beta}$-TUB, GAPDH, ${\beta}$-actin, B2m, G6pd2, Gyk, Gus, Hprt, Cyclophlin A, Tfrc, ${\alpha}$-tubulin and RPL13A) in the liver, stomach, small intestine, large intestine, kidney and spleen of mice fed GM or non-GM rice. We found no significant differences in the expression of housekeeping genes between the two groups of mice.

• ALGAE
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• 제31권2호
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• pp.167-174
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• 2016

Biological response of cells to variable conditions should affect the expression level of certain genes. Quantification of these changes in target genes needs stable internal controls. Real-time quantitative polymerase chain reaction (PCR) has traditionally used reference or ‘housekeeping’ genes, that are considered to maintain equal expression in different conditions, to evaluate changes in target genes between samples and experimental conditions. Recent studies showed that some housekeeping genes may vary considerably in certain biological samples. This has not been evaluated in red algae. In order to identify the optimal internal controls for real-time PCR, we studied the expression of eleven commonly used housekeeping genes; elongation factor 1-alpha, glyceraldehyde-3-phosphate dehydrogenase, β-actin, polyubiquitin, 30S ribosomal gene, 60S ribosomal gene, beta-tubulin, alpha-tubulin, translation initiation factor, ubiquitin-conjugating enzyme, and isocitrate dehydrogenase in different life-history stages of Bostrychia moritziana. Our results suggest that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and 30S ribosomal gene, have the most stable gene expression levels between the different life history stages (male, female, carposporophyte, and tetrasporophyte), while the other genes are not satisfactory as internal controls. These results suggest that the combinations of GAPDH and 30S would be useful as internal controls to assess expression level changes in genes that may control different physiological processes in this organism or that may change in different life history stages. These results may also be useful in other red algal systems.

• 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.

• 한국산학기술학회논문지
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• 제21권8호
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• pp.417-428
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• 2020

하우스키핑 유전자는 에너지생성, 물질합성, 세포사멸 및 세포방어 등과 같은 세포의 기본적인 기능을 수행하기 때문에 모든 유기체의 세포에서 발현된다. 세포의 기본적인 기능을 유지하기 때문에 발현 수준이 상대적으로 일정하여 단백질 발현 및 목적 유전자의 mRNA 발현 분석 등과 같은 유전자 발현 연구에서 기준 유전자로 사용되고 있다. 그러나 이들 유전자의 발현 수준은 조직과 세포마다 다를 수 있으며, 특정 환경 하에서 변할 수 있다. 그러므로 하우스키핑 유전자의 발현 안정성을 탐색하여 유전자 발현 연구에서 최적의 기준 유전자를 선택하는 것이 중요하다. 이 리뷰는 문헌을 통해 인간, 닭, 돼지 그리고 쥐에서 발견된 하우스키핑 유전자를 요약하고, geNorm, NormFinder 그리고 BestKeeper 소프트웨어를 통해 발현 안정성을 추정하였다. 하우스키핑 유전자의 발현 안정성에 대한 탐색은 유전자 발현 연구에서 실험 조건에 따라 가장 적합한 기준 유전자를 선별할 수 있고, 데이터의 정규화를 위해 적용될 수 있다.

• 한국어병학회지
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• 제22권3호
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• pp.391-400
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• 2009

Genetic identification of 17 fish-derived Aeromonas strains was attempted using 5 housekeeping genes. 16S rRNA, gyrB, rpoD, dnaJ and recA genes from the 17 strains were amplified, and total of 85 amplicons were sequenced. DNA sequences of the strains and type strains of the 17 Aeromonas homology groups were used for genetic identification and phylogenetic analyses. None of the strains was identified as a single species using the 16S rRNA gene, showing the same identities (average = 99.7%) with several Aeromonas species. According to gyrB, rpoD, dnaJ, and recA, 9 strains and RFAS-1 used in this study were identified as A. hydrophila and A. salmonicida, respectively. However, the other strains were closely related to 2 or more Aeromonas species (i.e., A. salmonicida, A. veronii, A. jandaei, A. media and A. troda) depending on the genetic marker used. In this study, gyrB, rpoD, dnaJ and recA gene sequences proved to be advantageous over 16S rRNA for the identification of field Aeromonas isolates obtained from fish. However, there are discrepancies between analyses of different phylogenetic markers, indicating there are still difficulties in genetic identification of the genus Aeromonas using the housekeeping genes used in this study. Advantages and disadvantages of each housekeeping gene should be taken into account when the gene is used for identification of Aeromonas species.

• 한국식품저장유통학회지
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• 제15권4호
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• pp.562-567
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• 2008

GM 및 non-GM 감자의 인체 위해성 여부를 판단하기 위하여 마우스 비장세포에서 인터루킨과 housekeeping gene의 발현을 RT-PCR로 비교 분석하였다. 유전자변형 감자의 잎과 이를 섭취한 마우스의 비장세포에서 발현되는 housekeeping gene의 발현을 수행한 결과, APRT, $\beta$-tubulin, Actin, Hsp 20.2, Cyclophilin, 18S RNA and Efla, and Tbp, GAPDH, $\beta$-actin, Tuba2, Hprt, Cyclophlin A, Tfrc, and RPL13A의 발현에는 유전자 변형 감자와 그렇지 않은 감자와의 차이를 발견할 수 없었다. GM작물의 안전성에 대한 연구는 많은 표본을 가지고, 보다 장기적인 관점에서 다양한 모니터링 시스템을 도입이 필요하다고 판단되며 이의 향후 연구에 많은 관심이 필요하다.

• Genomics & Informatics
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• 제11권4호
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• pp.272-276
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• 2013

Sequence analysis of the 16S rRNA gene has been widely used for the classification of microorganisms. However, we have been unable to clearly identify five Flavobacterium species isolated from a freshwater by using the gene as a single marker, because the evolutionary history is incomplete and the pace of DNA substitutions is relatively rapid in the bacteria. In this study, we tried to classify Flavobacterium species through multilocus sequence analysis (MLSA), which is a practical and reliable technique for the identification or classification of bacteria. The five Flavobacterium species isolated from freshwater and 37 other strains were classified based on six housekeeping genes: gyrB, dnaK, tuf, murG, atpA, and glyA. The genes were amplified by PCR and subjected to DNA sequencing. Based on the combined DNA sequence (4,412 bp) of the six housekeeping genes, we analyzed the phylogenetic relationship among the Flavobacterium species. The results indicated that MLSA, based on the six housekeeping genes, is a trustworthy method for the identification of closely related Flavobacterium species.

• 한국식품저장유통학회지
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• 제15권1호
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• pp.84-87
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• 2008

유전자 변형 작물은 생산성 측면에서 많은 장점이 있지만 이를 섭취할 경우 잠재적인 위험 요소들에 의해 많은 문제가 대두대고 있다. 본 연구는 저항성유전자를 이입한 배추에서 Profillin, Tubulin-${\alpha}$ (Tub-${\alpha}1$), Heat-shock protein (Bchsp 17.6) and Ubiquitin conjugating enzyme (UBE)의 발현과 이를 30일간 섭취한 마우스에서 ${\beta}$-actin(${\beta}$-act), ${\beta}$-2-microglobulin (B2m), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and ${\beta}$-glucuronidase (Gus)의 발현 정도를 RT-PCR을 통해 알아보았다. 실험 결과 저항성유전자를 이입한 배추와 그렇지 않은 배추의 유전자 발현 패턴은 큰 차이를 보이지 앓았으며, 이를 섭취한 마우스 장기에서도 발현에 따른 큰 차이는 나타나지 않았다.