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Reference Genes (reference + gene)

Distribution by Scientific Domains
Life Sciences70%
Medical Sciences30%

Selected Abstracts

Reference genes identified in the silkworm Bombyx mori during metamorphism based on oligonucleotide microarray and confirmed by qRT-PCR

INSECT SCIENCE, Issue 5 2008
Gen-Hong Wang
Abstract Gene expression quantification at mRNA level is very important for post-genomic studies, as gene expression level is the reflection of the special biological function of the target gene. Methods used for gene expression quantification, such as microarray or quantitative real-time polymerase chain reaction (qRT-PCR), require stable expressed reference genes. Thus, finding suitable control genes is essential for gene quantification. In this study, a genome-wide survey of reference genes during metamorphism was performed on silkworm Bombyx mori. Twelve genes were chosen as putative reference genes based on a whole genome oligonucleotide microarray normalized by external controls. Then, qRT-PCR was employed for further validation and selection of potential reference gene candidates. The results were analyzed, and stable genes were selected using geNorm 3.4 and NormFinder software. Finally, considering factors from every aspect, translation initiation factor 4A, translation initiation factor 3 subunit 4, and translation initiation factor 3 subunit 5 (represented by sw22934, swl4876, and swl3956) were selected as reliable internal controls across the examined developmental stages, while cytoplasmic actin (sw22671), the commonly used reference gene in a previous study was shown to vary drastically throughout the examined developmental stages. For future research, we recommend the use of the geometric mean of those three stable reference genes as an accurate normalization factor for data normalization of different developmental stages during metamorphism. [source]

Quantitative analysis of MUC1 and MUC5AC mRNA in pancreatic juice for preoperative diagnosis of pancreatic cancer

INTERNATIONAL JOURNAL OF CANCER, Issue 2 2006
Kenoki Ohuchida
Abstract Pancreatic juice is a promising type of diagnostic sample for pancreatic cancer, and members of the mucin (MUC) family are diagnostic candidates. To evaluate the utility of MUC family members as diagnostic markers, we measured MUC mRNA expression in pancreatic tissues and pancreatic juice obtained from patients with different pancreatic diseases as well as in pancreatic cancer cell lines by real-time PCR. Furthermore, to support the possibility of early diagnosis by quantification of MUC1 and MUC5AC, immunohistochemistry and microdissection-based quantitative analysis of mRNA were carried out. There was no significant correlation between MUC1 and MUC5AC expression in cell lines. When ,-actin was used as a reference gene, median MUC1 and MUC5AC mRNA expression levels were remarkably greater in tumoral tissues than in non-tumoral tissues, but median MUC4 and MUC6 mRNA expression levels were not. Receiver operating characteristic curve analysis showed that quantitative analysis of MUC1 and MUC5AC mRNA in pancreatic juice is better diagnostic modality than that of MUC4 and MUC6 mRNA. Immunohistochemistry showed that MUC1 and MUC5AC were highly expressed in invasive ductal carcinomas (IDC) and moderately expressed in high-grade pancreatic intraepithelial neoplasia (PanIN); no staining was observed in normal ducts. Analysis of cells isolated by microdissection showed stepwise upregulation of MUC1 and MUC5AC in the development of high-grade PanIN to IDC. Our results suggest that MUC1 and MUC5AC are upregulated stepwise in pancreatic carcinogenesis and that quantitative assessment of MUC1 and MUC5AC mRNA in pancreatic juice has high potential for preoperative diagnosis of pancreatic cancer. © 2005 Wiley-Liss, Inc. [source]

Nicotinic acetylcholine receptor-subunit mRNAs in the mouse superior cervical ganglion are regulated by development but not by deletion of distinct subunit genes

JOURNAL OF NEUROSCIENCE RESEARCH, Issue 5 2008
G. Putz
Abstract Mice with deletions of nicotinic ACh receptor (nAChR) subunit genes are valuable models for studying nAChR functions. We could previously show in the mouse superior cervical ganglion (SCG) that the absence of distinct subunits affects the functional properties of receptors. Here, we have addressed the question of whether deletions of the subunits ,5, ,7, or ,2 are compensated at the mRNA level, monitored by reverse transcription and quantitative real-time polymerase chain reaction. Relative to our reference gene, ,3, which is expressed in all SCG nAChRs, mRNA levels of ,4 showed little change from birth until adult ages in intact ganglia of wild-type mice. In contrast, ,4 declined sharply after birth and was barely detectable in adult animals. ,5, ,7, and ,2 subunit message levels also declined, though more slowly and less completely than ,4. The subunits ,6 and ,3 were detected by conventional polymerase chain reaction at very low levels, if at all, whereas ,2 was never seen in any of our samples. The developmental profile of nAChR mRNA levels in the three knockout strains did not differ markedly from that of wild-type mice. Likewise, message levels of nAChR subunits were similar in cultures prepared from either wild-type or knockout animals. Our observations indicate a developmental regulation of nAChR subunit mRNAs in the SCG of mice after birth that was not affected by the three knockouts under investigation. © 2007 Wiley-Liss, Inc. [source]

Selection of normalizer genes in conducting relative gene expression analysis of embryos

BIRTH DEFECTS RESEARCH, Issue 8 2003
Qin J. Zhang
Abstract BACKGROUND In relative gene expression analysis, a reference gene for sample normalization is required for determining target expression changes among experimental treatment groups. Since some developmental toxicants secondarily cause general growth retardation and/or other general biological changes, commonly used housekeeping genes may not serve as accurate normalizers. METHODS We conducted real-time polymerase chain reaction (PCR) with normalization to calculate relative target transcriptional change, using housekeeping and structure-specific expression genes as normalizers. Relative levels of Hoxb1 expression were measured in cultured rodent embryos at 24 hr post retinoic acid (RA) administration. Transcriptional response was also evaluated using two novel compounds that produced posterior axial and growth defects in rat whole-embryo culture. Embryos treated with these compounds were evaluated for general biological processes, and their respective biological states were considered in the context of the relative gene expression change calculated with the housekeeping normalizers. RESULTS Normalized RA-induced Hoxb1 expression demonstrated that only some reference genes accurately quantitated the expected 1.5- to 2-fold increase in Hoxb1 expression. Evaluation of the test compounds demonstrated that only normalization with the spatially-restricted hindbrain gene, Krox-20, calculated significant expression decreases of T -gene, a gene known to be functionally relevant in posterior axial development. Reduction in T -gene expression was confirmed qualitatively by whole-mount in situ hybridization. CONCLUSIONS Prudent reference gene selection is important in evaluating relative gene expression in embryos. An experimental control design is proposed to facilitate the identification of normalizing genes that will accurately calculate relative gene expression change in treated embryos. Birth Defects Research (Part A), 2003. © 2003 Wiley-Liss, Inc. [source]

Reference genes identified in the silkworm Bombyx mori during metamorphism based on oligonucleotide microarray and confirmed by qRT-PCR

INSECT SCIENCE, Issue 5 2008
Gen-Hong Wang
Abstract Gene expression quantification at mRNA level is very important for post-genomic studies, as gene expression level is the reflection of the special biological function of the target gene. Methods used for gene expression quantification, such as microarray or quantitative real-time polymerase chain reaction (qRT-PCR), require stable expressed reference genes. Thus, finding suitable control genes is essential for gene quantification. In this study, a genome-wide survey of reference genes during metamorphism was performed on silkworm Bombyx mori. Twelve genes were chosen as putative reference genes based on a whole genome oligonucleotide microarray normalized by external controls. Then, qRT-PCR was employed for further validation and selection of potential reference gene candidates. The results were analyzed, and stable genes were selected using geNorm 3.4 and NormFinder software. Finally, considering factors from every aspect, translation initiation factor 4A, translation initiation factor 3 subunit 4, and translation initiation factor 3 subunit 5 (represented by sw22934, swl4876, and swl3956) were selected as reliable internal controls across the examined developmental stages, while cytoplasmic actin (sw22671), the commonly used reference gene in a previous study was shown to vary drastically throughout the examined developmental stages. For future research, we recommend the use of the geometric mean of those three stable reference genes as an accurate normalization factor for data normalization of different developmental stages during metamorphism. [source]

Reference gene selection for real-time polymerase chain reaction in human lung cells subjected to cyclic mechanical strain

RESPIROLOGY, Issue 7 2008
Liao PINHU
Background and objective: The respiratory system is constantly exposed to mechanical forces that influence cellular phenotype in health and disease. Quantitative real-time PCR (qPCR) is widely used to determine gene expression. The validity of qPCR depends on using stable reference genes for normalization. The effect of cyclic mechanical strain on reference gene expression by lung epithelial, fibroblast and endothelial cells has not been studied systematically. Methods: The stability of expression of fourteen potential reference genes in response to six different regimens of cyclic mechanical strain was ranked using the geNorm tool in human lung epithelial cell lines (A549 and H441), human fetal lung fibroblasts (HFL-1), human lung microvascular endothelial cells, primary human lung fibroblasts and primary human alveolar type 2 (hAT2) cells. The expression variation of these reference genes was also screened in unstimulated whole human lung. Results: The stability of the selected reference genes varied within and between cell types, the variation in expression being greatest in primary cultures of hAT2. Correspondingly, the effect of expressing message for the stretch responsive gene IL-8 normalized to the 14 reference genes was greatest in the hAT2 cells, there being an almost fivefold difference in mRNA relative change comparing different reference genes in the same samples. The minimum number of genes required to derive a reliable normalization factor for experiments on single lung cell types undergoing mechanical strain was two and for whole human lung it was four. Conclusions: These results demonstrate that the optimal reference genes for lung cells subjected to CMS are cell type specific. [source]

Selection of normalizer genes in conducting relative gene expression analysis of embryos

BIRTH DEFECTS RESEARCH, Issue 8 2003
Qin J. Zhang
Abstract BACKGROUND In relative gene expression analysis, a reference gene for sample normalization is required for determining target expression changes among experimental treatment groups. Since some developmental toxicants secondarily cause general growth retardation and/or other general biological changes, commonly used housekeeping genes may not serve as accurate normalizers. METHODS We conducted real-time polymerase chain reaction (PCR) with normalization to calculate relative target transcriptional change, using housekeeping and structure-specific expression genes as normalizers. Relative levels of Hoxb1 expression were measured in cultured rodent embryos at 24 hr post retinoic acid (RA) administration. Transcriptional response was also evaluated using two novel compounds that produced posterior axial and growth defects in rat whole-embryo culture. Embryos treated with these compounds were evaluated for general biological processes, and their respective biological states were considered in the context of the relative gene expression change calculated with the housekeeping normalizers. RESULTS Normalized RA-induced Hoxb1 expression demonstrated that only some reference genes accurately quantitated the expected 1.5- to 2-fold increase in Hoxb1 expression. Evaluation of the test compounds demonstrated that only normalization with the spatially-restricted hindbrain gene, Krox-20, calculated significant expression decreases of T -gene, a gene known to be functionally relevant in posterior axial development. Reduction in T -gene expression was confirmed qualitatively by whole-mount in situ hybridization. CONCLUSIONS Prudent reference gene selection is important in evaluating relative gene expression in embryos. An experimental control design is proposed to facilitate the identification of normalizing genes that will accurately calculate relative gene expression change in treated embryos. Birth Defects Research (Part A), 2003. © 2003 Wiley-Liss, Inc. [source]