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

---

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

Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) is a technique with the potential of improving the quantification of disseminated epithelial cells (DEC) in haematological tissues due to its exquisite sensitivity. This sensitivity may lead to false positivity. Immunocytochemistry (ICC) is regarded as the standard methodology to diagnose DEC. In this study, detection with ICC was compared with quantitative real-time RT-PCR for CK-19 and mammaglobin (hMAM) mRNA in bone marrow (BM) of patients with metastatic breast cancer (MBC). Bone marrow was aspirated from 14 control patients and from 29 patients with MBC. Mononuclear cells (MNC) were isolated. Immunostaining was carried out with the Epimet kit. Quantitative PCR was performed on the ABI Prism 7700. The CK-19 and hMAM mRNA quantities were normalised against beta-Actin and calculated relative to a calibrator sample (relative gene expression). All controls were negative by ICC and for hMAM expression measured by RT-PCR, whereas the median RGE value for CK-19 was 0.57. For the MBC patients, the median RGE for hMAM was 0 and 10 out of 25 (40%) tested positive. Median RGE for CK-19 was 2.9 and 20 out of 25 (80%) tested positive. With ICC, the median value was 1 stained cell per sample, and 15 out of 24 (62%) samples were positive. A correlation was observed between CK-19 and hMAM expression (r=0.7; P=0.0003), and between hMAM expression and ICC (r=0.6; P=0.003). CK-19 expression and ICC (r=0.9; P<0.0001) showed the strongest correlation. Reverse transcriptase-polymerase chain reaction for CK-19 resulted in a higher number of positive BM samples of patients with MBC than ICC. Since an excellent correlation is observed between ICC and RT-PCR, and RT-PCR is probably more sensitive with the advantage of being less observer dependent and thus also more easy to automate, we consider our quantitative real-time RT-PCR method as validated for the detection of DEC in the bone marrow of breast cancer patients.
...
PMID:Real-time RT-PCR correlates with immunocytochemistry for the detection of disseminated epithelial cells in bone marrow aspirates of patients with breast cancer. 1550 29

Although human immunodeficiency virus type 1 (HIV-1) RNA is the acknowledged "gold standard" marker for monitoring disease activity in patients receiving highly active antiretroviral therapy (HAART), it remains unaffordable in resource-constrained settings. The present study investigated two commercially available kits for the detection of HIV-1 viral load markers as more affordable alternatives to HIV-1 RNA quantitation. The greatly improved heat-denatured, signal-boosted HiSens HIV-1 p24 Ag Ultra kit (Perkin-Elmer) and the ExaVir Load Quantitative HIV-RT kit (Cavidi Tech AB) were compared with the Amplicor HIV-1 Monitor (version 1.5) assay (Roche Molecular Systems Inc.). A total of 117 samples containing HIV-1 subtype C were analyzed by all three methodologies. Eighty-nine of these samples represented serial measurements from 20 patients receiving HAART. The remaining samples analyzed were from a group of treatment-naive patients. The association between the p24 antigen assay and the RNA assay was fairly strong (R(2) = 0.686). The association between the reverse transcriptase (RT) quantitation assay and the RNA assay was strong (R(2) = 0.810). Both alternative assays seemed most useful for the serial monitoring of patients receiving HAART (n = 89 plasma samples from 20 patients), as all assays showed a statistically significant downward trend over time, with the trend being either linear or curvilinear. In addition, all three assays showed negative correlations with the CD4 count (CD4 count versus RNA load, r = -0.336 and P = 0.001; CD4 count versus p24 antigen level, r = -0.541 and P < 0.0001; CD4 count versus RT level, r = -0.358 and P = 0.0006). Still of major concern are both the lack of sensitivity and the wide degrees of variability of both assays. However, both assays provide a less expensive alternative to the Roche viral load assay and demonstrate the same trends during treatment.
...
PMID:Evaluation of two commercially available, inexpensive alternative assays used for assessing viral load in a cohort of human immunodeficiency virus type 1 subtype C-infected patients from South Africa. 1569 92

To screen different combinations of prostate-specific membrane antigen (PSMA) promoter/enhancer with the strongest transcriptional activity in prostate-specific cells, we used PSMA regulatory elements to control specific expression of the target gene in gene therapy of prostate adenocarcinoma. PSMA promoter and enhancer DNA sequences were amplified from the LNCaP human prostate cancer cell line by polymerase chain reaction, then recombinant plasmids of the enhanced green fluorescent protein (EGFP: pEGFP-PSMA(Pro), pEGFP-PSMA(E-P), pEGFP-PSMA(E(r)-P), pEGFP-PSMA(E(d)-P), and pEGFP-PSMA(E(t)-P)) were constructed with molecular clonal techniques. At the same time, all experimental cell lines were analyzed for the expression of PSMA with the use of PSMA monoclonal antibody and the ABC immunohistochemical assay kit. After plasmids were transfected via liposome, we observed the expression of the reporter gene (EGFP) under a fluorescent microscope and compared the different levels of EGFP expression with reverse transcriptase polymerase chain reaction and flow cytometry so that we could choose the one with the highest transcriptional activity. Only the LNCaP cell line expressed PSMA positively with immunohistochemical stain. The PSMA promoter/enhancer had transcriptional activity in PSMA(+) cell lines and no activity in PSMA(-) cell lines. PSMA(E-P) achieved the strongest activity in different PSMA promoter/enhancer combinations. We confirmed the specific expression of PSMA in prostate cells again. Similarly, transcriptional activity of the PSMA promoter/enhancer was prostate specific. PSMA(E-P) achieved the strongest transcriptional activity among PSMA promoter/enhancer combinations, which could be used in advanced research for tissue-specific treatment.
...
PMID:Construction of prostate-specific expressed recombinant plasmids with high transcriptional activity of prostate-specific membrane antigen (PSMA) promoter/enhancer. 1571 27

We describe a combination of two established techniques for a novel application for constructing full-length cDNA clone libraries from environmental RNA. The cDNA was cloned without the use of prescribed primers that target specific genes, and the procedure did not involve random priming. Purified RNA was first modified by addition of a poly(A) tail and then was amplified by using a commercially available reverse transcriptase PCR (RT-PCR) cDNA synthesis kit. To demonstrate the feasibility of this approach, a cDNA clone library was constructed from size-fractionated RNA (targeting 16S rRNA) purified from a geothermally heated soil in Yellowstone National Park in Wyoming. The resulting cDNA library contained clones representing Bacteria and Eukarya taxa and several mRNAs. There was no exact clone match between this library and a separate cDNA library generated from an RT-PCR performed with unmodified rRNA and Bacteria-specific forward and universal reverse primers that were designed from cultivated organisms; however, both libraries contained representatives of the Firmicutes and the alpha-Proteobacteria. Unexpectedly, there were no Archaea clones in the library generated from poly(A)-modified RNA. Additional RT-PCRs performed with universal and Archaea-biased primers and unmodified RNA demonstrated the presence of novel Archaea in the soil. Experiments with pure cultures of Sulfolobus solfataricus and Halobacterium halobium revealed that some Archaea rRNA may not be a suitable substrate for the poly(A) tail modification step. The protocol described here demonstrates the feasibility of directly accessing prokaryote RNA (rRNA and/or mRNA) in environmental samples, but the results also illustrate potentially important problems.
...
PMID:Poly(A) polymerase modification and reverse transcriptase PCR amplification of environmental RNA. 1574 28

Despite viral contamination of recreational waters, only bacterial, not viral, indicators are monitored routinely, due to a lack of rapid and cost-effective assays. We used negatively charged filters to capture enteroviruses from seawater and freshwater. Viral RNA was extracted using a commercial kit, and the viruses were quantified by real-time quantitative reverse transcriptase PCR (qRT-PCR). Poliovirus (6.6 to 330,000 virus particles/ml) was added to samples from watersheds in Los Angeles, California, and analysis showed that with 50-ml samples, a cellulose acetate/nitrate (HA) filter yielded final recovery of 51% (r2= 0.99) in fresh water and 23% (r2= 0.90) in seawater. However, for additions of low levels of virus (more likely to represent field samples; <10(4) enterovirus particles/ml), the recovery was lower and more variable, with HA being best in freshwater (17%, r2= 0.97) and the type GF/F glass filter having higher average recovery in seawater (GF/F, 17%; r2= 0.93; HA 12%, r2= 0.87). The optimized method was used with 1-liter field samples from two very different freshwater "creeks" that drain into Santa Monica Bay, California: Topanga Creek (TC), a relatively pristine mountain creek, and Ballona Creek (BC), a concrete-lined urban storm drain. One TC site out of 10 and 2 BC sites out of 7 tested significantly positive for enteroviruses, with higher enterovirus concentrations in BC than in TC (ca. 10 to 25 versus 1 equivalent enterovirus particle/ml). The presented filtration-qRT-PCR approach is fast (<8 h from sampling to results), sensitive, and cost efficient and is promising for monitoring viral contamination in environmental water samples.
...
PMID:Rapid detection of enteroviruses in small volumes of natural waters by real-time quantitative reverse transcriptase PCR. 1608 45

Mast cells are crucial to the development of chronic allergic inflammation and are likely to play a critical role in host defense. In this chapter methodology for histamine and cytokine assays is provided. Crosslinkage of IgE receptor I (Fc epsilonRI) on cord blood-derived mast cells by myeloma IgE and anti-human IgE is used to induce histamine release. Histamine levels were measured in the culture supernatants using an enzyme-linked immunosorbent assay. A human mast cell line (HMC-1), derived from a patient with mast cell leukemia, was activated with interleukin (IL)-1beta to study cytokine production and gene expression. Cytokine gene expression was evaluated by reverse transcriptase polymerase chain reaction and cytokine production was assayed in culture supernatants using an enzyme-linked immunosorbent assay kit.
...
PMID:Mast cell histamine and cytokine assays. 1611 Jan 60

Telomerase, a ribonucleoprotein enzyme that functions as a reverse transcriptase, is detected exclusively in immortal cells such as germ cells, stem cells and cancer cells. Telomerase activity is present in almost all human cancers. Telomerase activation is considered to be essential to maintain the integrity of the replicating tumor cell and to establish immortality. Based on this concept antiestrogen should initially regulate estrogen-stimulated telomerase but the enzyme would be expected to be constitutive in tamoxifen-resistant tumor cells. We have studied the estrogen regulation of telomerase in T47D:A18 breast cancer cells with a TRAPEZE Telomerase detection kit. Estradiol significantly increased telomerase activity after a 2-day treatment. Telomerase activity induced by estradiol was up to 10-fold higher within 4 days. Antiestrogens 4-hydroxytamoxifen (4-OHT) and ICI 182,780 were inactive alone and significantly blocked estradiol-stimulated increase in telomerase. These effects were correlated with changes in cell replications and changes in the cell cycle. In contrast, 4-OHT resistant T47D:A18 cells (T47D:A18/4-OHT, cultured in 1 microM 4-OHT for 6 months) grew spontaneously and had no changes in the cell cycle with estrogen treatment. The estrogen receptor (ERalpha) was present and still regulated at an estrogen responsive luciferase reporter gene with estrogen despite the fact that progesterone receptor was not increased in response to estradiol in T47D:A18/4-OHT cells. However, telomerase activity was increased about 40-fold in T47D:A18/4-OHT cells and this was not regulated by ICI 182,780. We conclude that the differential regulation of telomerase gene might be an important transition for tamoxifen resistance in T47D:A18 breast cancer cells.
...
PMID:Deregulation of estrogen induced telomerase activity in tamoxifen-resistant breast cancer cells. 1621 Dec 43

The suitability of a reverse transcriptase (RT) PCR assay was evaluated for the detection of bovine central nervous system (CNS) tissue specifically in liver sausages. Because of its emulsifying effect, CNS tissue was frequently added to this kind of meat product in the past. On standard samples, the RT-PCR technique reliably detected a concentration of 0.25% bovine CNS tissue in liver sausages stored for up to 28 days. Following the successful application of RT-PCR for the detection of bovine CNS tissue in these specially prepared samples, a field study was performed with a total of 258 liver sausages purchased in retail markets. All sausages were tested with both an RT-PCR assay and a commercial enzyme-linked immunosorbent assay (ELISA) kit. Nine (3.5%) of the retail liver sausage samples were positive for CNS tissue in the ELISA, but none were positive for this tissue in the RT-PCR assay. All positive ELISA results indicated the presence of 0.23 to 0.30% CNS tissue. Recent studies have indicated that the RT-PCR assay is not as sensitive for porcine CNS tissue as for bovine CNS tissue, which this assay can detect at 0.25%. Although the ELISA is not species specific, the CNS tissue detected by the ELISA is assumed to stem from a nonbovine species. The RT-PCR technique is a sensitive tool for the detection of bovine CNS tissues in a problematic matrix such as liver sausage. ELISA screening followed by a species-specific RT-PCR assay for bovine CNS tissue is a practical approach for monitoring meat products for compliance with European food regulations.
...
PMID:Detection of bovine central nervous system tissue in liver sausages using a reverse transcriptase PCR technique and a commercial enzyme-linked immunosorbent assay. 1624 26

The present work was to study induction of cytochrome P450 (CYP)3A and CYP2H1 gene by reverse transcriptase polymerase chain reaction (RT-PCR) and quantitative RTPCR in Bantam, Bantamized White Leghorn and White Leghorn chicks. Out of 18 chicks total 3 from each group (Bantam, Bantamized White Leghorn and White Leghorn) were treated intraperitoneal with phenobarbital at the dose rate of 12 mg/100 g (body weight) while the control group was treated with the saline. Total RNA was extracted from the liver samples using Tri Reagent based method. First strand cDNA was synthesized using one step RT-PCR kit. The PCR was performed and the product was subjected to agarose gel electrophoresis. Quantitative RT-PCR was conducted to quantify gene expression level of CYP3A and CYP2H1 genes. Relative expression ratio of CYP3A and CYP2H1 genes was calculated using relative expression software tool (REST). It was found that CYP3A is up regulated by factor of 1.34, 14.51 and 1.00 in Bantam, Bantamized White Leghorn and White Leghorn chicks, respectively. In Bantam and Bantamized White Leghorn chicks CYP2H1 gene was up regulated by factor 1.50 and 80.87, respectively but down regulated by a factor of 1.97 in White Leghorn chicks. The PCR efficiency ranged from 1.30 to 1.70, 0.86 to 1.70 and 0.91 to 1.58 for CYP3A, CYP2H1 and beta-actin, respectively in Bantam, Bantamized White Leghorn and White Leghorn chicks.
...
PMID:Comparative evaluation of phenobarbital-induced CYP3A and CYP2H1 gene expression by quantitative RT-PCR in Bantam, Bantamized White Leghorn and White Leghorn chicks. 1629 89

The TruGene human immunodeficiency virus type 1 (HIV-1) genotyping kit/OpenGene DNA sequencing system (Bayer HealthCare, Tarrytown, NY) reliably produced clinically acceptable resistance profiles for reverse transcriptase and protease inhibitors on patient samples diluted to approximately 100 copies/ml following extraction with the QIAamp viral RNA minikit (QIAGEN Inc., Valencia, CA). One modification of the standard protocol was made to guarantee PCR amplification: a centrifugation step to concentrate virus was added before RNA extraction. For genotypic antiretroviral resistance testing, no significant differences in the identification and sensitivity of detection for codon mutations, base mutations, and multibase sites were found between the original and diluted samples.
...
PMID:Performance of the TruGene human immunodeficiency virus type 1 genotyping kit and OpenGene DNA sequencing system on clinical samples diluted to approximately 100 copies per milliliter. 1646 31


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>

---