Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.2.1.13 (glyceraldehyde-3-phosphate dehydrogenase)
6,511 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Simplex optimization has generated several media that have improved the development of mouse preimplantation embryos in vitro. One objective of this study was to compare the development of preimplantation mouse embryos in one of these computer-optimized media, KSOM, with embryos that developed in vivo, in terms of the relative abundances of specific mRNAs involved in metabolism, transcription, and cell proliferation. First, however, since studies have indicated an improvement of other simple embryo culture media by addition of amino acids, the effects of the addition of amino acids to KSOM (KSOM/AA) on preimplantation development were assessed. We find that addition of both essential and nonessential amino acids to KSOM augments development in vitro, as compared to development supported by KSOM without amino acids. This augmentation is observed starting at the blastocyst stage, and is associated with increased rate of development to the blastocyst stage, increased frequency of hatching, and increased number of cells in the blastocysts. Reverse-transcription PCR was then used to assess the relative abundance of mRNAs for actin, glyceraldehyde-3-phosphate dehydrogenase, Na+, K(+)-ATPase, Sp1, TATA box-binding protein TBP, IGF-I, IGF-II, IGF-I receptor, and IGF-II receptor in embryos that developed in vivo and in vitro using KSOM/AA. Eight out of 9 of these mRNAs were present in the 8-cell embryos and blastocysts raised in KSOM/AA in amounts that were indistinguishable from those in embryos that developed in vivo. It is concluded that KSOM/AA provides an environment in which preimplantation mouse embryos can undergo development that is quantitatively similar to that occurring in vivo.
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PMID:Preimplantation development of mouse embryos in KSOM: augmentation by amino acids and analysis of gene expression. 765 76

Real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) is a powerful method for measurement of gene expression for diagnostic and prognostic studies of non-Hodgkin's lymphomas (NHL). In order for this technique to gain wide applicability, it is critically important to establish a uniform method for normalization of RNA input. In this study, we have determined the best method to quantify the RNA/cDNA input per reaction and searched for the most useful endogenous control genes for normalization of the measurements, based on their abundance and lowest variability between different types of lymphoid cells. To accomplish these aims, we have analyzed the RNA expression of 11 potential endogenous control genes (glyceraldehyde-3-phosphate dehydrogenase, beta-actin, peptidylprolyl isomerase A, beta 2 microglobulin, protein kinase cGMP-dependent, type I, hypoxanthine phosphoribosyltransferase 1, TATA box binding protein, transferrin receptor, large ribosomal protein, beta-glucoronidase and 18S ribosomal RNA). In all, 12 different B- and T-cell lymphoma/leukemia cell lines, 80 B- and T-cell NHL specimens, and resting and activated normal B and T lymphocytes were screened. Normalization of the nucleic acid input by spectrophotometric OD(260) measurement of RNA proved more reliable than spectrophotometric or fluorometric measurements of cDNA or than electrophoretic estimation of the ribosomal and mRNA fractions. The protein kinase cGMP-dependent, type I (PRKG1) and the TBP genes were expressed at common abundance and exhibited the lowest variability among the cell specimens. We suggest that for further lymphoma studies based on the real-time RT-PCR quantification of gene expression, that RNA input in each reaction be equalized between the specimens by spectrophotometric OD(260) measurements. The expression of the gene of interest in different samples should be normalized by concomitant measurement of the PRKG1 and/or the TBP gene products.
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PMID:Optimization of quantitative real-time RT-PCR parameters for the study of lymphoid malignancies. 1268 39

Preeclampsia and diabetes are complications of pregnancy that contribute to maternal and perinatal mortality worldwide. Results emerging from molecular studies of placentae may elucidate etiologically important genomic alterations. Appropriate application of real time reverse transcription (RT) PCR in comparative gene expression studies requires endogenous housekeeping genes to normalize between sample variations. Ideal housekeeping genes must have stable tissue expression, but few have been specifically studied in the placenta. We sought to identify candidate control genes by analyzing seven functionally distinct housekeeping genes (B2M, GAPDH, HMBS, HPRT, SDHA, TBP, YWHAZ) for their expression stability and level in the placenta. mRNA isolated from 20 placentae was analyzed for gene expression using RT-PCR. Expression stability (M) was assessed using normalization strategies previously used for other tissues. TBP and SDHA were the most stable, with an average expression stability of M = 0.43, followed by YWHAZ (M = 0.44) > HPRT (M = 0.53) > HMBS (M = 0.57) > GAPDH (M = 0.61) > B2M (M = 0.69). The genes tested ranged in abundance, with an approximately 300-fold increase from the lowest (HMBS) to the highest (B2M). By using TBP, SDHA and YWHAZ, with greater expression stability than those housekeeping genes commonly used in placenta studies, gene expression profile comparisons will have more sensitivity and specificity.
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PMID:Evaluation of housekeeping genes in placental comparative expression studies. 1608 39

The liver has an intrinsic ability to undergo active proliferation and recover functional liver mass in response to an injury response. This regenerative process involves a complex yet well orchestrated change in the gene expression profile. To produce accurate and reliable gene expression of target genes during various stages of liver regeneration, the determination of internal control housekeeping genes (HKGs) those are uniformly expressed is required. In the present study, the gene expression of 8 commonly used HKGs, including GAPDH, ACTB, HPRT1, GUSB, PPIA, TBP, TFRC, and RPL4, were studied using mouse livers that were quiescent and actively regenerating induced by partial hepatectomy. The amplification of the HKGs was statistically analyzed by two different mathematical algorithms, geNorm and NormFinder. Using this method, PPIA and TBP gene expression found to be relatively stable regardless of the stages of liver regeneration and would be ideal for normalization to target gene expression.
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PMID:Reference gene selection for real-time RT-PCR in regenerating mouse livers. 1860 71

Comparative gene expression studies in the placenta may provide insights into molecular mechanisms of important genomic alterations in pregnancy disorders. Endogenous reference genes often referred to as housekeeping genes, are routinely used to normalise gene expression levels. For this reason, it is important that these genes be empirically evaluated for stability between placental samples including samples from complicated pregnancies. To address this issue, six candidate housekeeping genes including several commonly used ones (ACTB, GAPDH, 18S rRNA, TBP, SDHA and YWHAZ) were investigated for their expression stability in placentae obtained from pregnancies complicated by idiopathic FGR (n=25) and gestation-matched control pregnancies (n=25). Real-time PCR was performed using pre-validated gene expression assay kits. The geNorm program was used for gene stability measure (M) for the entire housekeeping genes in all control and FGR-affected placental samples. Results showed that GAPDH and 18S rRNA were most stable, with an average expression stability of M=0.441 and 0.443, respectively, followed by YWHAZ (M=0.472). SDHA, ACTB and TBP were the least stable housekeeping genes (M=0.495, 0.548 and 1.737, respectively). We recommend geometric averaging of two or more housekeeping genes to determine relative gene expression levels between FGR-affected and control placentae.
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PMID:GAPDH, 18S rRNA and YWHAZ are suitable endogenous reference genes for relative gene expression studies in placental tissues from human idiopathic fetal growth restriction. 1868 3

Careful validation of reference genes used for the normalization of real-time RT-PCR data is required to obtain accurate results regarding gene expression. We evaluated the stability of seven commonly used reference genes in the cerebral cortex and hippocampus of rats 3 days following traumatic brain injury (TBI). HPRT, SDHA, and GUSB were found to be the most stable reference genes in the cerebral cortex, whereas B2MG, TBP, and GAPDH were the most stable in the hippocampus. The use of three reference genes was determined to be the optimal number for accurate normalization of data. To illustrate this point, when our gene of interest, substance P (SP), was normalized against the three most stable reference genes in both brain areas, we found no significant difference between injured and uninjured rats at the 3-day time point. However, when our SP data were normalized to each reference gene individually, SP mRNA level was highly variable depending on the reference gene chosen. The results of the present study highlight the importance of validating reference genes to be used for real-time RT-PCR analysis. The use of the most stable reference genes presented here will allow more accurate normalization of gene expression data in TBI.
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PMID:Validation of reference genes for normalization of real-time quantitative RT-PCR data in traumatic brain injury. 1871 51

Expression patterns of candidate genes with important functions in animal metabolism can help to identify potential molecular markers for cattle production traits. Reverse transcription followed by polymerase chain reaction is a method for rapid and accurate mRNA quantification. However, for exact comparison of mRNA quantity in various samples or tissues, it is important to choose appropriate reference genes. In cattle, little information is available on the expression stability of housekeeping genes (HKGs). The aim of the present study is to develop a set of reference genes that can be used for normalization of concentrations of mRNAs of genes expressed in the bovine liver, kidney, pituitary and thyroid. The study was performed on 6-, 9-, and 12-month-old bulls of dairy and meat cattle breeds. Six HKGs were investigated: ACTB, GAPDH, HPRTI, SDHA, TBP, and YWHAZ. The most stably expressed potential reference HKGs differed among tissues/organs examined: ACTB, TBP, YWHAZ, GAPDH, HPRTI, and SDHA in the liver; GAPDH and YWHAZ in the kidney; GAPDH and SDHA in the pituitary; and TBP and HPRTI in the thyroid. The results showed that the use of a single gene for normalization may lead to relatively large errors, so it is important to use multiple control genes based on a survey of potential reference genes applied to representative samples from specific experimental conditions.
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PMID:Evaluation of reference genes for studies of gene expression in the bovine liver, kidney, pituitary, and thyroid. 1902 84

Quantitative gene expression measurements from tumor tissue are frequently compared with matched normal and/or adjacent tumor tissue expression for diagnostic marker gene selection as well as assessment of the degree of transcriptional deregulation in cancer. Selection of an appropriate reference gene (RG) or an RG panel, which varies depending on cancer type, molecular subtypes, and the normal tissues used for interindividual calibration, is crucial for the accurate quantification of gene expression. Several RG panels have been suggested in breast cancer for making comparisons among tumor subtypes, cell lines, and benign/malignant tumors. In this study, expression patterns of 15 widely used endogenous RGs (ACTB, TBP, GAPDH, SDHA, HPRT, HMBS, B2M, PPIA, GUSB, YWHAZ2, PGK1, RPLP0, PUM1, MRPL19, and RPL41), and three candidate genes that were selected through analysis of two independent microarray datasets (IL22RA1, TC22, ZNF224) were determined in 23 primary breast tumors and their matched normal tissues using qRT-PCR. Additionally, 18S rRNA, ACTB, and SDHA were tested using randomly primed cDNAs from 13 breast tumor pairs to assess the rRNA/mRNA ratio. The tumors exhibited significantly lower rRNA/mRNA ratio when compared to their normals, on average. The expression of the studied RGs in breast tumors did not exhibit differences in terms of grade, ER, or PR status. The stability of RGs was examined based on two different statistical models, namely GeNorm and NormFinder. Among the 18 tested endogenous reference genes, ACTB and SDHA were identified as the most suitable reference genes for the normalization of qRT-PCR data in the analysis of normal matched tumor breast tissue pairs by both programs. In addition, the expression of the gelsolin (GSN) gene, a well-known downregulated target in breast tumors, was analyzed using the two most suitable genes and different RG combinations to validate their effectiveness as a normalization factor (NF). The GSN expression of the tumors used in this study was significantly lower than that of normals showing the effectivity of using ACTB and SDHA as suitable RGs in this set of tumor-normal tissue panel. The combinational use of the best performing two RGs (ACTB and SDHA) as a normalization factor can be recommended to minimize sample variability and to increase the accuracy and resolution of gene expression normalization in tumor-normal paired breast cancer qRT-PCR studies.
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PMID:Identification of endogenous reference genes for qRT-PCR analysis in normal matched breast tumor tissues. 1954 72

Quantitative real-time RT-PCR (RT-qPCR) has proven to be a valuable molecular technique in gene expression quantification. Target gene expression levels are usually normalized to a stably expressed reference gene simultaneously determined in the same sample. It is critical to select optimal reference genes to interpret data generated by RT-qPCR. However, no suitable reference genes have been identified in human ovarian cancer to date. In this study, 10 housekeeping genes, ACTB, ALAS1, GAPDH, GUSB, HPRT1, PBGD, PPIA, PUM1, RPL29, and TBP as well as 18S rRNA that were already used in various studies were analyzed to determine their applicability. Totally 20 serous ovarian cancer specimens and 20 normal ovarian epithelial tissue specimens were examined. All candidate reference genes showed significant differences in expression between malignant and nonmalignant groups except GUSB, PPIA, and TBP. The expression stability and suitability of the 11 genes were validated employing geNorm and NormFinder. GUSB, PPIA, and TBP were demonstrated as the most stable reference genes and thus could be used as reference genes for normalization in gene profiling studies of serous ovarian cancer, while the combination of two genes (GUSB and PPIA) or the all three genes should be recommended as a much more reliable normalization strategy.
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PMID:Identification of suitable reference genes for gene expression studies of human serous ovarian cancer by real-time polymerase chain reaction. 1962 37

Gene expression studies in intestinal epithelial and stromal cells are a common tool for investigating the mechanisms by which the homeostasis of the small intestine is regulated under normal and pathological conditions. Quantitative real-time PCR (qPCR) is a sensitive and highly reproducible method of gene expression analysis, with expression levels quantified by normalization against reference genes in most cases. However, the lack of suitable reference genes for epithelial cells with different differentiation states and nonepithelial tissue cells has limited the application of qPCR in gene expression studies of small intestinal samples. In this study, 13 housekeeping genes, ACTB, B2M, GAPDH, GUSB, HPRT1, HMBS, HSP90AB1, RPL13A, RPS29, RPLP0,PPIA, TBP, and TUBA1, were analyzed to determine their applicability for isolated crypt cells, villus cells, deepithelialized mucosa, and whole mucosa of the mouse small intestine. Using geNorm and NormFinder software, GUSB and TBP were identified as the most stably expressed genes, whereas the expressions of the commonly used reference genes GAPDH, B2M, and ACTB, and ribosomal protein genes RPL13A, RPS29, and RPLP0 were relatively unstable. Thus, this study demonstrates that GUSB and TBP are the optimal reference genes for the normalization of gene expression in the mouse small intestine.
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PMID:Normalizing genes for real-time polymerase chain reaction in epithelial and nonepithelial cells of mouse small intestine. 2003 9


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