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:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We previously showed that Wilms tumor gene (WT1) expression level, measured by quantitative reverse transcriptase polymerase chain reaction (RT-PCR), was useful as an indicator of minimal residual disease (MRD) in leukemia and myelodysplastic syndrome. However, in conventional quantitative RT-PCR (CQ-PCR), RT-PCR must be performed for various numbers of cycles depending on WT1 expression level. In the present study, we developed a new real-time quantitative RT-PCR (RQ-PCR) method for quantitating WT1 transcripts. Results of intraassay and interassay variability tests demonstrated that the real-time WT1 assay had high reproducibility. WT1 expression levels measured by the RQ- and the CQ-PCR methods were strongly correlated (r = 0.998). Furthermore, a strong correlation was observed among WT1 transcript values normalized with 3 different control genes (beta-actin, ABL, and glyceraldehyde-3-phosphate dehydrogenase) and between relative WT1 transcript values with WT1 expression in K562 cells as the reference and absolute WT1 transcript copy numbers per microgram RNA. When WT1 expression and minor bcr-abl expression were concurrently monitored in 2 patients with bcr-abl-positive acute lymphoblastic leukemia, both MRDs changed mostly in parallel, indicating the reliability and validity of our RQ-PCR method. In conclusion, this RQ-PCR method is convenient and reliable for monitoring MRD and enables routine clinical use of a WT1 assay.
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PMID:Monitoring minimal residual disease in leukemia using real-time quantitative polymerase chain reaction for Wilms tumor gene (WT1). 1468 94

The accuracy of 18S rRNA, beta-actin mRNA and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA as indicators of cell number when used for normalization in gene expression analysis of T lymphocytes at different activation stages was investigated. Quantitative real-time reverse transcriptase-polymerase chain reaction was used to determine the expression level of 18S rRNA, beta-actin mRNA, GAPDH mRNA and mRNA for six cytokines in carefully counted samples of resting human peripheral blood mononuclear cells (PBMCs), intestinal lymphocytes and PBMCs subjected to polyclonal T-cell activation. The 18S rRNA level in activated and resting PBMCs and intestinal lymphocytes was essentially the same, while the levels of beta-actin and GAPDH mRNAs fluctuated markedly upon activation. When isolated gammadeltaTCR(+), CD4(+) and CD8(+) subpopulations were studied, 18S rRNA levels remained unchanged after 21 h of activation but increased slightly after 96 h. In contrast, there was a 30-70-fold increase of GAPDH mRNA/cell in these cell populations upon activation. Cytokine analysis revealed that only normalization to 18S rRNA gave a result that satisfactorily reflected their mRNA expression levels per cell. In conclusion, 18S rRNA was the most stable housekeeping gene and hence superior for normalization in comparative analyses of mRNA expression levels in human T lymphocytes.
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PMID:Utility of the housekeeping genes 18S rRNA, beta-actin and glyceraldehyde-3-phosphate-dehydrogenase for normalization in real-time quantitative reverse transcriptase-polymerase chain reaction analysis of gene expression in human T lymphocytes. 1518 52

Survivin has been identified as one of the top 4 transcripts among 3.5 million human transcriptomes uniformly up-regulated in cancer tissues but not in normal tissues. Therefore, we quantitatively determined the messenger RNA (mRNA) expression profile for survivin by a real-time reverse transcriptase-polymerase chain reaction (RT-PCR) technique in 113 patients with leukemias, such as adult T-cell leukemia (ATL), acute lymphoid leukemia (ALL), acute myeloid leukemia (AML), chronic myeloid leukemia in crisis, and chronic lymphocytic leukemia (CLL), and in 25 cell lines, including 7 ATL cell lines and 15 solid-tumor cell lines. Furthermore, we examined whether the plasma level of survivin protein as measured by enzyme-linked immunosorbent assay (ELISA) substituted for mRNA expression by PCR quantification. Gene expression was quantitatively confirmed to be up-regulated in approximately 90% of ATL and acute leukemia cases and in all of the cell lines tested, whereas it was down-regulated in almost all cases of CLL. Furthermore, with respect to the interpretation of the gene expression findings, attention was paid to standardization with a housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), in the real-time PCR quantification, because the variability in GAPDH expression among the different cell types was significant. GAPDH expression was relatively low in ATL cells and high in ALL and AML cells. The rates of increase in the levels of survivin protein in the plasma of ATL patients and in the supernatants from in vitro cultures of solid-tumor cell lines were low compared with rates of increase of the mRNA and protein level in the cells, suggesting that the protein levels in plasma do not always reflect survivin expression in tumor cells. Our findings indicate the potential clinical relevance of survivin quantified by real-time PCR but not for the protein level in plasma as determined by ELISA, especially in cases of ATL and acute leukemias.
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PMID:Clinical relevance of survivin as a biomarker in neoplasms, especially in adult T-cell leukemias and acute leukemias. 1529 68

A rapid analysis method for murine endothelin-A (ETA) and endothelin-B (ETB) receptor gene expression levels was established using real-time quantitative reverse transcriptase-polymerase chain reaction. We designed primer pairs and TaqMan probes specific for the two cDNAs and available for mouse and rat systems. The standard curve method was used to examine relative expression. The gene expression levels of ETA and ETB were estimated as gene expression rates by normalizing to the expression of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase. To examine the reproducibility of this assay system, we calculated the intra-assay and interassay coefficients of variation of the gene expression rate and found that a greater than 1.6-fold increase in relative gene expression is detectable as a significant change. ETA and ETB receptor gene expression was found in all 16 organs of mouse and rat examined, and high levels of expression were observed in the lung, uterus, ovary, intestine, and cerebellum. The gene expression patterns essentially agreed with those determined by RNase protection assay, Northern blot, and conventional endpoint polymerase chain reaction. These results show that this new rapid, sensitive, and semi-automated method is accurate, quantitative, and reproducible. This method is also useful for examining regulation of hormone receptor gene expression under physiological conditions in organs.
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PMID:Real-time polymerase chain reaction quantification of gene expression levels of murine endothelin-A and endothelin-B receptors: gene expression profiles by the standard curve method. 1583 11

Several studies have shown that specific mRNA sequences can be successfully detected in formalin-fixed, paraffin-embedded tissues using reverse transcriptase-polymerase chain reaction (RT-PCR). Here, we test the hypothesis that gene expression levels can be accurately quantified in formalin-fixed, paraffin-embedded tissues by determining the ratio between the copy number of the mRNA molecule of interest and the mRNA copy number of a so-called housekeeping gene. The mRNA copy numbers of the variably expressed multiple drug resistance gene (MDR)-1 and four housekeeping genes (hypoxanthine phosphoribosyl-transferase-1, glyceraldehyde-3-phosphate dehydrogenase, beta-actin, and elongation factor-1a) were quantified by real-time-quantitative RT-PCR before and after formalin-fixation and paraffin-embedding of 576 tissue samples (heart, kidney, spleen, liver) from three beagle dogs. The results indicate that fixation and embedding drastically altered the ratios between the different mRNA copy numbers and that the relative expression levels of MDR-1 per any of the housekeeping genes were artificially increased or decreased up to more than tenfold. It would thus appear questionable to normalize quantitative expression data from fixed and embedded tissues by using housekeeping genes as reference. In contrast, tissue autolysis of up to 24 h and long-term storage of embedded tissues of up to 20 years had no additional effects.
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PMID:Impact of formalin-fixation and paraffin-embedding on the ratio between mRNA copy numbers of differently expressed genes. 1604 95

The pathogenesis of inflammatory bowel disease (IBD) and antibiotic-responsive diarrhea (ARD) in dogs likely involves an interaction between the intestinal immune system and luminal bacterial or food antigens. German Shepherd Dogs (GSD) are particularly predisposed to both IBD and ARD. CD4+ T cells are important for the regulation of immune responses in the mucosa, and they exert their effects through the secretion of cytokines. The present study examined the role of cytokines in the pathogenesis of canine chronic enteropathies by quantification of mRNA encoding interleukin-2 (IL-2), IL-4, IL-5, IL-6, IL-10, IL-12, IL-18, interferon gamma, tumor necrosis factor-alpha, transforming growth factor-beta, and glyceraldehyde-3-phosphate dehydrogenase by real-time reverse transcriptase polymerase chain reaction in duodenal mucosal biopsies obtained from 39 dogs with chronic diarrhea and 18 control dogs. Contemporaneously collected biopsies were assessed for histologic changes with a 4-point grading system. No significant difference in the expression of cytokine mRNA (P > .01) was detected between dogs with and those without chronic diarrhea. Similarly, no significant differences in cytokine mRNA expression were observed between GSD and other breeds with chronic diarrhea, or between histologically normal duodenal mucosa and that with evidence of inflammatory change. Failure to detect a difference in mRNA expression does not rule out the possibility of a defect downstream at the level of translation or protein function. No conclusion can be drawn from these data as to the predominant CD4+ cell type in the pathogenesis of these canine chronic enteropathies.
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PMID:Cytokine mRNA quantification in duodenal mucosa from dogs with chronic enteropathies by real-time reverse transcriptase polymerase chain reaction. 1623 8

Tissue engineering of articular cartilage usually requires the isolation and culture of chondrocytes. Previous studies have suggested that enzymatic isolation may alter the metabolic activity and growth rate of chondrocytes. This study examined the effects of 4 common isolation protocols on chondrocyte gene expression, morphology, and total cell yield immediately following the digest (t = 0) and after 2 culture periods (24 h and 1 week). Cartilage explants were digested using 1 of 4 protocols: (1) 6-h collagenase digest, (2) 22-h collagenase digest, (3) 45-min trypsin digest followed by a 3-h collagenase digest, or (4) 1.5-h pronase digest followed by a 3-h collagenase digest. Gene expression levels for glyceraldehyde-3-phosphate dehydrogenase, type I collagen, type II collagen, aggrecan, superficial zone protein, matrix metalloproteinase- 1, and tissue inhibitor of metalloproteinase-1 were measured at t = 0 h, 24 h, and 1 week using quantitative reverse transcriptase-polymerase chain reaction. In this study, cell yield was greatest for the 22-h collagenase and pronase-collagenase digests. However, the data indicate that a 6-h collagenase digest has the fewest gene expression changes compared to native cells. For tissue engineering, data from this study suggest that when cell yield is critical, a 22-h collagenase digest is preferable, but when obtaining cells closest to native chondrocytes is more desired, the 6-h collagenase digest is more beneficial.
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PMID:The effects of isolation on chondrocyte gene expression. 1699 90

A subtracted cDNA library was used to identify specific genes that increase in post-mortem muscle of rainbow trout (Oncorhynchus mykiss) during on-ice storage. Of the 200 cDNAs analyzed, 82 had significant homologies to previously identified genes from salmonids and other species such as homologues of troponin I, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and so on, whereas 40 had no significant homologies and were designated as unknown. Comparison of gene expression profiles by dot blot hybridization confirmed an increase or induction of mRNA in the muscle after 3 h of on-ice storage compared to that at 0 h after death. Real-time reverse transcriptase-Polymerase Chain Reaction analysis showed that troponin I and GAPDH mRNAs were increased by 24 h and, in particular, that the change in troponin I mRNA was greater than that of GAPDH mRNA. These results suggest that the increased mRNAs in rainbow trout muscle occurred by transcription immediately after death.
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PMID:Post-mortem changes in gene expression of the muscle tissue of rainbow trout, Oncorhynchus mykiss. 1714 27

Our group has shown that mechanical stimulation increases the stiffness of stem cell-collagen sponge constructs at 14 days in culture and subsequent rabbit patellar tendon repairs at 12 weeks postsurgery. What remains unclear is which genes might be responsible for this increase in stiffness. Therefore, the objective of this study was to determine how a tensile stimulus affects the gene expression of stem cell-collagen sponge constructs used to repair rabbit central patellar tendon defects. Tissue-engineered constructs were created by seeding mesenchymal stem cells (MSCs) from 10 adult rabbits at 0.14 x 10(6) cells/construct in type I collagen sponges. Half of the constructs were mechanically stimulated once every 5 min for 8 h/d to a peak strain of 2.4% for 2 weeks. The other half remained in an incubator without mechanical stimulation for 2 weeks. After 14 days in culture, half of the stimulated and nonstimulated constructs were prepared to determine the expression of collagen type I, collagen type III, decorin, fibronectin, and glyceraldehyde-3-phosphate dehydrogenase genes using real-time quantitative reverse transcriptase polymerase chain reaction. The remaining constructs were mechanically tested to determine their mechanical properties. Two weeks of in vitro mechanical stimulation significantly increased collagen type I and collagen type III gene expression of the stem cell-collagen sponge constructs. Stimulated constructs showed 3 and 4 times greater collagen type I (p = 0.0001) and collagen type III gene expression (p = 0.001) than nonstimulated controls. Stimulated constructs also had 2.5 times the linear stiffness and 4 times the linear modulus of nonstimulated constructs. However, mechanical stimulation did not significantly increase decorin or fibronectin gene expression (p = 0.2) after 14 days in culture. This study shows that mechanical stimulation of cell-sponge constructs produces similar increases in the expression of 2 structural genes, as well as linear stiffness and linear modulus.
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PMID:Mechanical stimulation increases collagen type I and collagen type III gene expression of stem cell-collagen sponge constructs for patellar tendon repair. 1751 15

The quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) is a sensitive and very efficient technique for quantification of gene expression. However, qRT-PCR relies on accurate normalization of gene expression data, as RNA recovery and cDNA synthesis efficiency might vary from sample to sample. In the present study, six putative reference genes were validated for normalization of gene expression in three different tissues and in white blood cells from pigs experimentally infected with the common respiratory pathogen Actinobacillus pleuropneumoniae. Two dedicated validation programs (geNorm and Normfinder) were used to rank the six reference genes from best to worst. qRT-PCR data for the proinflammatory cytokine IL-6 was normalized using the proposed genes from geNorm and Normfinder as well as the commonly used reference gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). IL-6 expression was quantified in white blood cells, liver, lymph nodes and tonsils from 10 infected pigs and 5 control pigs. After normalization using either geNorm or Normfinder IL-6 was shown to be significantly up-regulated (P<0.05) in all of the tissues from infected animals compared to non-infected control animals with a good agreement of expression differences between the two programs. On the contrary, normalization of IL-6 expression data from blood using GAPDH rendered the difference between infected and non-infected groups non-significant, and resulted in significantly different values compared to geNorm (P=0.01). Based on these results, we recommend to validate putative reference genes before normalization.
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PMID:Validation of putative reference genes for qRT-PCR normalization in tissues and blood from pigs infected with Actinobacillus pleuropneumoniae. 1754 55


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