• Genomics & Informatics
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• v.2 no.2
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• pp.92-98
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• 2004

A cDNA microarray experiment is one of the most useful high-throughput experiments in medical informatics for monitoring gene expression levels. Statistical analysis with a cDNA microarray medical data requires a normalization procedure to reduce the systematic errors that are impossible to control by the experimental conditions. Despite the variety of normalization methods, this. paper suggests a more general and synthetic normalization algorithm with a control gene set based on previous studies of normalization. Iterative normalization method was used to select and include a new control gene set among the whole genes iteratively at every step of the normalization calculation initiated with the housekeeping genes. The objective of this iterative normalization was to maintain the pattern of the original data and to keep the gene expression levels stable. Spatial plots, M&A (ratio and average values of the intensity) plots and box plots showed a convergence to zero of the mean across all genes graphically after applying our iterative normalization. The practicability of the algorithm was demonstrated by applying our method to the data for the human photo aging study.

• Asian-Australasian Journal of Animal Sciences
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• v.21 no.11
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• pp.1673-1679
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• 2008

In order to study the biological function of gapdh gene in yak, and prove whether the gapdh gene was a useful intra-reference gene that can be given an important role in molecular biology research of yak, the cDNA sequence encoding glyceraldehyde-3-phosphate dehydrogenase from yak was cloned by the RT-PCR method using gene specific PCR primers. The sequence results indicated that the cloned cDNA fragment (1,008 bp) contained a 1,002 bp open reading frame, encoding 333 amino acids (AAs) with a molecular mass of 35.753 kDa. The deduced amino acids sequence showed a high level of sequence identity to Bos Taurus (99.70%), Xenopus laevis (94.29%), Homo sapiens (97.01%), Mus musculus (97.90%) and Sus scrofa (98.20%). The expression of yak's gapdh gene in heart, spleen, kidney and brain tissues was also detected; the results showed that the gapdh gene was expressed in all these tissues. Further analysis of yak GAPDH amino acid sequence implied that it contained a complete glyceraldehyde-3-phosphate dehydrogenase active site (ASCTTNCL) which ranged from 148 to 155 amino acid residues. It also contained two conserved domains, a NAD binding domain in its N-terminal and a complete catalytic domain of sugar transport in its C-terminal. The phylogenetic analysis showed that yak and Bos taurus were the closest species. The prediction of secondary structures indicated that GAPDH of yak had a similar secondary structure to other isolated GAPDH. The results of this study suggested that the gapdh gene of yak was similar to other species and could be used as the intra-reference to analyze the expression of other genes in yak.

• Biomedical Science Letters
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• v.24 no.3
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• pp.221-229
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• 2018

Trichophyton rubrum is one of the well-known pathogenic fungi and causes dermatophytosis and cutaneous mycosis in human world widely. However, there are not an available sequence type (ST) classification methods and previous studies for T. rubrum until now. Therefore, currently, molecular biological tools using their DNA sequences are used for genotype identification and classification. In the present study, in order to characterize the genetic diversity and the phylogenetic relation of T. rubrum clinical isolates, five different housekeeping genes, such as actin (ACT), calmodulin (CAL), RNA polymerase II (RPB2), superoxide dismutase 2 (SOD2), and ${\beta}$-tubulin (BT2) were analyzed using by multilocus sequence typing (MLST). Also, DNA sequence analysis was performed to examine the differences between the sequences of Trichophyton strains and the identified genetic variations sequence. As a result, most of the sequences were shown to have highly matched rates in their housekeeping genes. However, genetic variations were found on three different positions of ${\beta}$-tubulin gene and were shown to have changed from $C{\rightarrow}G$ (1766), $G{\rightarrow}T$ (1876), and $C{\rightarrow}A$ (1886). To confirm the association with T. rubrum inheritance, a phylogenetic tree analysis was performed. It was classified as four clusters, but there was little significant correlation. Even so, MLST analysis is believed to be helpful for determining the genetic variations of T. rubrum in cases where there is more large-scale data accumulation. In conclusion, the present study demonstrated the first MLST analysis of T. rubrum in Korea and explored the possibility that MLST could be a useful tool for studying the epidemiology and evolution of T. rubrum through further studies.

• The Plant Pathology Journal
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• v.27 no.4
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• pp.301-309
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• 2011

The filamentous fungus Fusarium graminearum is an important cereal pathogen. Although quantitative realtime PCR (qRT-PCR) is commonly used to analyze the expression of important fungal genes, no detailed validation of reference genes for the normalization of qRT-PCR data has been performed in this fungus. Here, we evaluated 15 candidate genes as references, including those previously described as housekeeping genes and those selected from the whole transcriptome sequencing data. By a combination of three statistical algorithms (BestKeeper, geNorm, and NormFinder), the variation in the expression of these genes was assessed under different culture conditions that favored mycelial growth, sexual development, and trichothecene mycotoxin production. When favoring mycelial growth, GzFLO and GzUBH expression were most stable in complete medium. Both EF1A and GzRPS16 expression were relatively stable under all conditions on carrot agar, including mycelial growth and the subsequent perithecial induction stage. These two genes were also most stable during trichothecene production. For the combined data set, GzUBH and EF1A were selected as the most stable. Thus, these genes are suitable reference genes for accurate normalization of qRT-PCR data for gene expression analyses of F. graminearum and other related fungi.

• Biomedical Science Letters
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• v.29 no.4
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• pp.220-230
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• 2023

Identification of the pathogens causing infection is important in terms of patient's health management and infection control. Synovial fluids could be used as clinical samples to detect causative pathogens of prosthetic joint infections (PJIs) using molecular diagnostic assays, therefore, normalization and validation of clinical samples are necessary. Microbial culture is considered the gold standard for all infections, including PJIs. Recently, molecular diagnostic methods have been developed to overcome the limitation of microbial culture. Therefore, guideline for validating clinical samples to provide reliable results of molecular diagnostic assays for infectious diseases is required in clinical field. The present study aimed to develop an accurate validating method of synovial fluid clinical samples using GAPDH gene as an internal control to perform the quantitative PCR TaqMan probe assay to detect pathogens causing PJIs.

• Journal of Microbiology and Biotechnology
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• v.21 no.4
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• pp.421-429
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• 2011

For precise identification of a Lactobacillus K1 isolate, LC-MS/MS analysis of the putative surface layer protein was performed. The results obtained from LTQ-FT-ICR mass spectrometry confirmed that the analyzed protein spot is the surface layer protein originating from Lb. helveticus species. Moreover, the identified protein has the highest similarity with the surface layer protein from Lb. helveticus R0052. To evaluate the proteomic study, multilocus sequence analysis of selected housekeeping gene sequences was performed. Combination of 16S rRNA sequencing with partial sequences for the genes encoding the RNA polymerase alpha subunit (rpoA), phenylalanyl-tRNA synthase alpha subunit (pheS), translational elongation factor Tu (tuf), and Hsp60 chaperonins (groEL) also allowed to classify the analyzed isolate as Lb. helveticus. Further classification at the strain level was achieved by sequencing of the slp gene. This gene showed 99.8% identity with the corresponding slp gene of Lb. helveticus R0052, which is in good agreement with data obtained by nano-HPLC coupled to an LTQ-FT-ICR mass spectrometer. Finally, LC-MS/MS analysis of surface layer proteins extracted from three other Lactobacillus strains proved that the proposed method is the appropriate molecular tool for the identification of S-layer-possessing lactobacilli at the species and even strain levels.

• Fisheries and Aquatic Sciences
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• v.19 no.4
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• pp.21.1-21.11
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• 2016

Background: The evaluation of suitable reference genes as normalization controls is a prerequisite requirement for launching quantitative reverse transcription-PCR (RT-qPCR)-based expression study. In order to select the stable reference genes in abalone Haliotis discus hannai tissues (gill and hepatopancreas) under heavy metal exposure conditions (Cu, Zn, and Cd), 12 potential candidate housekeeping genes were subjected to expression stability based on the comprehensive ranking while integrating four different statistical algorithms (geNorm, NormFinder, BestKeeper, and ${\Delta}CT$ method). Results: Expression stability in the gill subset was determined as RPL7 > RPL8 > ACTB > RPL3 > PPIB > RPL7A > EF1A > RPL4 > GAPDH > RPL5 > UBE2 > B-TU. On the other hand, the ranking in the subset for hepatopancreas was RPL7 > RPL3 > RPL8 > ACTB > RPL4 > EF1A > RPL5 > RPL7A > B-TU > UBE2 > PPIB > GAPDH. The pairwise variation assessed by the geNorm program indicates that two reference genes could be sufficient for accurate normalization in both gill and hepatopancreas subsets. Overall, both gill and hepatopancreas subsets recommended ribosomal protein genes (particularly RPL7) as stable references, whereas traditional housekeepers such as ${\beta}-tubulin$ (B-TU) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were ranked as unstable genes. The validation of reference gene selection was confirmed with the quantitative assay of MT transcripts. Conclusions: The present analysis showed the importance of validating reference genes with multiple algorithmic approaches to select genes that are truly stable. Our results indicate that expression stability of a given reference gene could not always have consensus across tissue types. The data from this study could be a good guide for the future design of RT-qPCR studies with respect to metal regulation/detoxification and other related physiologies in this abalone species.

• Journal of Microbiology and Biotechnology
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• v.34 no.3
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• pp.570-579
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• 2024

Root-nodule nitrogen-fixing bacteria are known for being specific to particular legumes. This study isolated the endophytic root-nodule bacteria from the nodules of legumes and examined them to determine whether they could be used to promote the formation of nodules in other legumes. Forty-six isolates were collected from five leguminous plants and screened for housekeeping (16S rRNA), nitrogen fixation (nifH), and nodulation (nodC) genes. Based on the 16S rRNA gene sequencing and phylogenetic analysis, the bacterial isolates WC15, WC16, WC24, and GM5 were identified as Rhizobium, Sphingomonas, Methylobacterium, and Bradyrhizobium, respectively. The four isolates were found to have the nifH gene, and the study confirmed that one isolate (GM5) had both the nifH and nodC genes. The Salkowski method was used to measure the isolated bacteria for their capacity to produce phytohormone indole acetic acid (IAA). Additional experiments were performed to examine the effect of the isolated bacteria on root morphology and nodulation. Among the four tested isolates, both WC24 and GM5 induced nodulation in Glycine max. The gene expression studies revealed that GM5 had a higher expression of the nifH gene. The existence and expression of the nitrogen-fixing genes implied that the tested strain had the ability to fix the atmospheric nitrogen. These findings demonstrated that a nitrogen-fixing bacterium, Methylobacterium (WC24), isolated from a Trifolium repens, induced the formation of root nodules in non-host leguminous plants (Glycine max). This suggested the potential application of these rhizobia as biofertilizer. Further studies are required to verify the N₂-fixing efficiency of the isolates.

• Korean Journal of Clinical Laboratory Science
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• v.50 no.3
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• pp.331-336
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• 2018

In this study, multi-locus sequence typing (MLST) analysis of 40 clinically isolated Candida albicans in tertiary hospitals in Daejeon, Korea, confirmed the nucleotide sequence and phylogenetic relationships of the strains collected from different specimen sources. The general variations found in seven different housekeeping genes of C. albicans, collected from urine and sputum, peripheral blood, central line blood, and other specimens, were analyzed. The phylogenetic tree was divided into 18 sub-clusters (1), a central line blood (2), others (5), sputum (1), peripheral blood (6), sputum (1), and urine (1), and the isolates at the same site were confirmed to have genetic similarity. Consequently, genetic similarity and the potential relevance were found in the strains collected from the same specimen sources. MLST analysis of C. albicans suggests that persistent data accumulation of phylogenetic gene variations of C. albicans may help establish infectious disease studies and epidemiological surveillance systems.

• BMB Reports
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• v.40 no.2
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• pp.277-285
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• 2007

PCD (programmed cell death) is important mechanism for development, homeostasis and disease. To analyze the gene expression pattern in brain cells undergoing PCD in response to serum deprivation, we analyzed the cDNA microarray consisting of 2,300 genes and 7 housekeeping genes of cortical cells derived from mouse embryonic brain. Cortical cells were induced apoptosis by serum deprivation for 8 hours. We identified 69 up-regulated genes and 21 down-regulated genes in apoptotic cells. Based on the cDNA microarray data four genes were selected and analyzed by RT-PCR and northern blotting. To characterize the role of UNC-51-like kinase (ULK2) gene in PCD, we investigated cell death effect by ULK2. And we examined expression of several genes that related with PCD. Especially GAPDH was increased by ULK2. Theses findings indicated that ULK2 is involved in apoptosis through p53 pathway.