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Reverse Transcription PCR (reverse + transcription_pcr)
Kinds of Reverse Transcription PCR Terms modified by Reverse Transcription PCR Selected AbstractsORIGINAL ARTICLE: Selective Downregulation of Phosphoinositide 3-Kinase alpha in Leukocytes During PregnancyAMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY, Issue 2 2009Anne Rohrbach Problem, During pregnancy, it is crucially important that the mother's immune system tolerates the developing embryo. Although a number of mechanisms of immunological tolerance have been described, little is known about intracellular signaling events, causing a decrease in the mother's leukocyte activity. Method of study, We investigated the expression and activity of phosphoinositide 3-kinases (PI3K) in maternal blood cells of healthy volunteers by Reverse Transcription PCR and Western blotting. Results, Our data reveal a selective downregulation of the p110, catalytic isoform. This correlated with a slight decrease in PI3K activity as judged by the levels of phosphorylated Akt. Conclusion, As PI3K are involved in signal transduction of various leukocyte receptors, this downregulation may comprise a means of holding immune functions at bay. [source] Prothrombin kringle-2 induces death of mesencephalic dopaminergic neurons in vivo and in vitro via microglial activationJOURNAL OF NEUROSCIENCE RESEARCH, Issue 7 2010Sang Ryong Kim Abstract We have shown that prothrombin kringle-2 (pKr-2), a domain of human prothrombin distinct from thrombin could activate cultured rat brain microglia in vitro. However, little is known whether pKr-2-induced microglial activation could cause neurotoxicity on dopaminergic (DA) neurons in vivo. To address this question, pKr-2 was injected into the rat substantia nigra (SN). Tyrosine hydroxylase (TH) immunohistochemistry experiments demonstrate significant loss of DA neurons seven days after injection of pKr-2. In parallel, pKr-2-activated microglia were detected in the SN with OX-42 and OX-6 immunohistochemistry. Reverse transcription PCR and double-label immunohistochemistry revealed that activated microglia in vivo exhibit early and transient expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and several proinflammatory cytokines. The pKr-2-induced loss of SN DA neurons was partially inhibited by the NOS inhibitor NG -nitro-L-arginine methyl ester hydrochloride, and the COX-2 inhibitor DuP-697. Extracellular signal-regulated kinase 1/2, c-Jun N-terminal kinase and p38 mitogen-activated protein kinase were activated in the SN as early as 1 hr after pKr-2 injection, and localized within microglia. Inhibition of these kinases led to attenuation of mRNA expression of iNOS, COX-2 and several proinflammatory cytokines, and rescue of DA neurons in the SN. Intriguingly, following treatment with pKr-2 in vitro, neurotoxicity was detected exclusively in co-cultures of mesencephalic neurons and microglia, but not microglia-free neuron-enriched mesencephalic cultures, indicating that microglia are required for pKr-2 neurotoxicity. Our results strongly suggest that microglia activated by endogenous compound(s), such as pKr-2, are implicated in the DA neuronal cell death in the SN. © 2009 Wiley-Liss, Inc. [source] Dynein light chain family in Tetrahymena thermophilaCYTOSKELETON, Issue 2 2007David E. Wilkes Abstract Dyneins are large protein complexes that produce directed movement on microtubules. In situ, dyneins comprise combinations of heavy, intermediate, light-intermediate, and light chains. The light chains regulate the locations and activities of dyneins but their functions are not completely understood. We have searched the recently sequenced Tetrahymena thermophila macronuclear genome to describe the entire family of dynein light chains expressed in this organism. We identified fourteen genes encoding putative dynein light chains and seven genes encoding light chain-like proteins. RNA-directed PCR revealed that all 21 genes were expressed. Quantitative real time reverse transcription PCR showed that many of these genes were upregulated after deciliation, indicating that these proteins are present in cilia. Using the nomenclature developed in Chlamydomonas, Tetrahymena expresses two isoforms each of LC2, LC4, LC7, and Tctex1, three isoforms of p28, and six LC8/LC8-like isoforms. Tetrahymena also expresses two LC3-like genes. No Tetrahymena orthologue was found for Chlamydomonas LC5 or LC6. This study provides a complete description of the different genes and isoforms of the dynein light chains that are expressed in Tetrahymena, a model organism in which the targeted manipulation of genes is straightforward. Cell Motil. Cytoskeleton 2006. © 2006 Wiley-Liss, Inc. [source] Single nucleotide polymorphisms 5, upstream the coding region of the NEIL2 gene influence gene transcription levels and alter levels of genetic damageGENES, CHROMOSOMES AND CANCER, Issue 11 2008Carla J. Kinslow NEIL2 (EC 4.2.99.18), a mammalian DNA glycosylase and ortholog of the bacterial Fpg/Nei, excises oxidized DNA lesions from bubble or single-stranded structures, suggesting its involvement in transcription-coupled DNA repair. Because base excision repair (BER) proteins act collectively and in a progressive fashion, their proper balance is essential for optimal repair. Thus, inter-individual variability in transcription levels of NEIL2 may predispose to compromised DNA repair capacity and genomic instability by altering the balance of critical BER proteins. In a study of lymphocytes of 129 healthy subjects, using absolute quantitative reverse transcription PCR, we found that NEIL2 transcription varied significantly (up to 63 fold) and that this variability was influenced by certain single nucleotide polymorphisms (SNPs) located 5, of the start site. Using the mutagen sensitivity assay to characterize the biological significance of these SNPs, we observed a significant increase in mutagen-induced genetic damage associated with two SNPs in the promoter region of the NEIL2 gene. To characterize the functional significance of these SNPs, we engineered luciferase-reporter constructs of the NEIL2 promotor with mutations corresponding to these SNPs. We transfected these constructs into MRC-5 cells and evaluated their impact on NEIL2 expression levels. Our results indicate that NEIL2 expression was significantly reduced by over 50% (P < 0.01) in the presence of two SNPs, ss74800505 and rs8191518, located near the NEIL2 start site, which were in significant linkage disequilibrium (D, = 73%; P < 0.05). This first report on in vivo variability in NEIL2 expression in humans identifies SNPs in the NEIL2 promoter region that have functional effects. © 2008 Wiley-Liss, Inc. [source] Androgen profiles among Egyptian adults considering liver statusJOURNAL OF GASTROENTEROLOGY AND HEPATOLOGY, Issue 7pt2 2008Cristina E Aguilar Abstract Background and aim:, Hepatitis C virus (HCV) and environmental hepatotoxins may have an indirect influence on health by altering the synthesis and function of hormones, particularly reproductive hormones. We aimed to evaluate liver diseases and sex steroid hormones in Egypt, which has the highest prevalence of HCV worldwide. Methods:, We measured markers of hepatitis B virus (HBV), HCV and schistosomiasis infection as well as liver function in 159 apparently healthy subjects. We measured total testosterone (T), sex-hormone binding globulin (SHBG) and albumin, and calculated the free androgen index. Results:, Anti-HCV antibodies were detected in 51% of men and 42% of women. Based on HCV reverse transcription PCR (RT-PCR) of 44 men and 33 women, 11% of men and 21% of women showed HCV viremia. There was schistosomiasis in 25% of men and 9% of women, and mixed HCV viremia and schistosomiasis in 57% of men and 52% of women. Compared with men with schistosomiasis only (mean 593.3 ± 73.4 ng/dL), T was higher in men with mixed HCV viremia and schistosomiasis (mean 854.5 ± 47.9 ng/dL; P = 0.006) and men with mixed chronic HCV and schistosomiasis (mean 812.1 ± 43.3 ng/dL; P = 0.001). Men with mixed chronic HCV and schistosomiasis had also significantly higher SHBG (mean 57.7 ± 3.9 ng/dL) than males with schistosomiasis only (mean 34.8 ± SE 4.5 ng/dL; P = 0.0003). Conclusion:, Future investigations should consider that a high prevalence of asymptomatic liver disease may alter associations between hormone concentrations and chronic disease etiology. [source] Distinct C-terminus of the B subunit of factor XIII in a population-associated major phenotype: the first case of complete allele-specific alternative splicing products in the coagulation and fibrinolytic systemsJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 7 2009H. IWATA Summary.,Objectives: The purpose of this study was to elucidate the molecular bases of the heterogeneity of the B subunit of coagulation factor XIII (FXIII-B), classified by isoelectric focusing into its three population-associated major phenotypes. Methods and Results: By genetic sequencing and polymerase chain reaction (PCR),restriction fragment length polymorphism analyses, a C-to-G change was identified in intron K for the Asian-associated major phenotype FXIII-B*3. A transcript containing the novel exon XII, was detected by reverse transcription PCR using hepatocyte cell lines with this allele. The exclusive existence of a novel C-terminal peptide in a homozygote of FXIII-B*3 was also detected by matrix-assisted laser-desorption ionization time of flight mass spectrometry. The FXIII-B*3 isoform had a C-terminus 15 residues longer than the other isoforms, containing two additional basic amino acids and one extra acidic amino acid. Accordingly, the C-to-G nucleotide substitution created an efficient splice acceptor AG dinucleotide, which resulted in allele-specific alternative splicing in intron K. When compared with FXIII-B*1, the third major phenotype, FXIII-B*2, had an A-to-G change in exon III, converting His95 to Arg, and a rare phenotype, FXIII-B*4, had an A-to-T change in exon VII, converting Glu368 to Val. Conclusions: We found an extremely rare event of complete allele-specific alternative splicing for FXIII-B. The FXIII-B*3 isoform had a distinct C-terminal peptide, while the FXIII-B*2 and FXIII-B*4 isoforms had His95 to Arg and Glu368 to Val substitutions, respectively, which led to differential isoelectric points of these isoforms. Such variations in the amino acid sequence of FXIII-B may have profound effects on its structure,function relationship, plasma FXIII levels, and disease susceptibility. [source] Selective amplification of bacterial RNA: use of a DNA primer containing mismatched bases near its 3, terminus to reduce false-positive signalsLETTERS IN APPLIED MICROBIOLOGY, Issue 3 2000K. Koo A reverse transcription PCR (RT,PCR) method designed to reduce false-positive results due to the co-amplification of contaminating genomic DNA is reported. Feasibility of the method was evaluated using 16S rRNA sequences specific to Bacillus cereus. A DNA oligonucleotide primer, consisting of 22-bases containing three consecutive mismatched bases near its 3, terminus (primer B16RT), was used for reverse transcription and in subsequent cDNA amplification. Specific rRNA was reverse transcribed at low temperature (40 °C or 45 °C) in the presence of primer B16RT. As subsequent PCR using primer B16RT at high (62 °C) annealing temperatures is not nearly as efficient as amplification using the specific primer, amplification of genomic DNA was hindered relative to the amplification of cDNA. The method was readily adapted to the selective amplification of mRNA of the Listeria monocytogenes listeriolysin O (hly) gene. [source] Deciphering regulatory mechanisms for secondary metabolite production in the myxobacterium Sorangium cellulosum So ce56MOLECULAR MICROBIOLOGY, Issue 6 2007Shwan Rachid Summary Sorangium cellulosum strains produce approximately 50% of the biologically active secondary metabolites known from myxobacteria. These metabolites include several compounds of biotechnological importance such as the epothilones and chivosazols, which, respectively, stabilize the tubulin and actin skeletons of eukaryotic cells. S. cellulosum is characterized by its slow growth rate, and natural products are typically produced in low yield. In this study, biomagnetic bead separation of promoter-binding proteins and subsequent inactivation experiments were employed to identify the chivosazol regulator, ChiR, as a positive regulator of chivosazol biosynthesis in the genome-sequenced strain So ce56. Overexpression of chiR under the control of T7A1 promoter in a merodiploid mutant resulted in fivefold overproduction of chivosazol in a kinetic shake flask experiment, and 2.5-fold overproduction by fermentation. Using quantitative reverse transcription PCR and gel shift experiments employing heterologously expressed ChiR, we have shown that transcription of the chivosazol biosynthetic genes (chiA,chiF) is directly controlled by this protein. In addition, we have demonstrated that ChiR serves as a pleiotropic regulator in S. cellulosum, because mutant strains lack the ability to develop into regular fruiting bodies. [source] Quantitative analysis of messenger RNA abundance for ribosomal protein L-15, cyclophilin-A, phosphoglycerokinase, ,-glucuronidase, glyceraldehyde 3-phosphate dehydrogenase, ,-actin, and histone H2A during bovine oocyte maturation and early embryogenesis in vitroMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 3 2006AnilKumar Bettegowda Abstract Real-time reverse transcription PCR has greatly improved the ease and sensitivity of quantitative gene expression studies. However, measurement of gene expression generally requires selection of a valid reference (housekeeping gene) for data normalization to compensate for inherent variations. Given the dynamic nature of early embryonic development, application of this technology to studies of oocyte and early embryonic development is further complicated due to limited amounts of starting material and a paucity of information on constitutively expressed genes for data normalization. We have validated quantitative procedures for real-time reverse transcription polymerase chain reaction (RT-PCR) analysis of mRNA abundance during bovine meiotic maturation and early embryogenesis and utilized this technology to determine temporal changes in mRNA abundance for ribosomal protein L-15, cyclophilin-A, phosphoglycerokinase, ,-glucuronidase, glyceraldehyde-3-phosphate dehydrogenase, ,-actin, and histone H2A. Quantification of amounts of specific exogenous RNAs added to samples revealed acceptable rates of RNA recovery and efficiency of reverse transcription with minimal variation. Progression of bovine oocytes to metaphase II resulted in reduced abundance of polyadenylated, but not total transcripts for majority of above genes; however phosphoglycerokinase exhibited a significant decline in both RNA populations. Abundance of mRNAs for above genes in early embryos generally remained low until the blastocyst stage, but abundance of ribosomal protein L-15 mRNA was increased at the morula stage and histone H2A mRNA showed dynamic changes prior to embryonic genome activation. Results demonstrate a valid approach for quantitative analysis of mRNA abundance in oocytes and embryos, but do not support constitutive expression of above genes during early embryonic development. Mol. Reprod. Dev. © 2005 Wiley-Liss, Inc. [source] A functional polymorphism in Pre-miR-146a gene is associated with prostate cancer risk and mature miR-146a expression in vivo,THE PROSTATE, Issue 5 2010Bin Xu Abstract BACKGROUND A G,>,C polymorphism (rs2910164) which is located in the sequence of miR-146a precursor, results in a change from a G:U pair to a C:U mismatch in its stem region. To explore whether rs2910164 plays any role in prostate cancer (CaP), we analyzed the association between miR-146a polymorphism and risk of CaP and the expression of miR-146a with different genotypes in CaP tissues in southern Chinese Han population. MATERIALS AND METHODS Two hundred fifty-one CaP and 280 control subjects were included in the cancer association study, and 15 CaP tissue samples were used to test the expression of the miRNA precursors by real-time quantitative reverse transcription PCR. RESULTS We found that subjects carrying CC homozygotes had a 0.65-fold reduced risk (95% CI,=,0.43,0.99) than those carrying GG/GC genotypes (P,=,0.03), and the C allele displayed a lower prevalence of CaP compared with the G allele (OR,=,0.73, 95% CI,=,0.57,0.94, P,=,0.01). Moreover, hsa-miR-146a quantification showed that homozygous carriers of the C-variant had significantly decreased miRNA levels compared to the carriers of the GG/GC genotype. CONCLUSIONS The natural genetic variation in pre-miR-146a affects the amount of mature miR-146a, contributes to the genetic predisposition to CaP. Prostate 70: 467,472, 2010. © 2009 Wiley-Liss, Inc. [source] Detection of viruses in nasal swab samples from horses with acute, febrile, respiratory disease using virus isolation, polymerase chain reaction and serologyAUSTRALIAN VETERINARY JOURNAL, Issue 1-2 2007K Dynon Objective To examine the association of viruses with acute febrile respiratory disease in horses. Design Nasal swab and serum samples were collected from 20 horses with acute febrile upper respiratory disease that was clinically assessed to have a viral origin. Methods Each of the samples was inoculated onto equine fetal kidney, RK13 and Vero cell cultures, and viral nucleic acid was extracted for polymerase chain reaction (PCR) or reverse transcription PCR. PCR primers were designed to amplify nucleic acid from viruses known to cause or be associated with acute febrile respiratory disease in horses in Australia. A type specific ELISA was used to measure equine herpesvirus (EHV1 and EHV4) antibody, and serum neutralisation assays were used to measure equine rhinitis A virus (ERAV) and equine rhinitis B virus 1 and 2 (ERBV1 and ERBV2) antibody titres in serum samples. Results Virus was isolated from 4 of 20 nasal swab samples. There were three isolations of EHV4 and one of ERBV2. By PCR, virus was identified in the nasal swab samples of 12 of the 20 horses. Of those that were positive, 17 viruses were detected as follows: one triple positive (EHV4, EHV2, and EHV5), three double positives (EHV4, ERBV and EHV2, EHV5 in two horses) and eight single positives (EHV4 in two horses, EHV2 in one horse, EHV5 in six horses and ERBV in six horses). Conclusion By virus isolation and PCR, 17 viruses were identified in nasal swab samples from 12 of 20 horses that had acute febrile respiratory disease consistent with a diagnosis of virus infection. Initial PCR identification and subsequent virus isolation led to the isolation of ERBV2 for the first time in Australia and the second time anywhere of ERBV2. [source] No evidence of intrauterine transmission of hepatitis A virus from a mother to a premature infantACTA PAEDIATRICA, Issue 10 2009Bo Selander Abstract Aim:, To determine whether or not an intrauterine transmission of hepatitis A virus (HAV) occurred from an infected mother to her premature infant delivered by caesarean section. Methods:, The mother and her child were tested for HAV by serology and reverse transcription PCR. Results:, An outbreak of HAV infection was seen among children and a 33-year-old day-care teacher, pregnant in third trimester, at a day-care centre in southern Sweden. Due to premature labour and diminished foetal movements a caesarean section was performed and a premature girl in gestational weeks 33 + 1 was born. During the 3-week postnatal hospitalization period the child presented no clinical symptoms of HAV infection and anti-HAV IgM antibodies remained undetectable at day 14 and 109 after birth. Furthermore HAV RNA remained undetectable by reverse transcription PCR in the child's blood at birth and in throat and faeces for the first 3 and 4 weeks of life respectively. HAV RNA in the mother's blood was detected at 6 days prior to and at 17 days after delivery. HAV RNA was undetectable in breast milk when tested on day 3 after delivery. Conclusion:, There was no evidence of intrauterine transmission of hepatitis A virus from a viraemic mother to her premature child delivered at gestational week 33 + 1 by caesarean section. [source] Immunoreactivity profile of peripheral blood mononuclear cells from patients with ragweed-induced allergic rhinitisCLINICAL & EXPERIMENTAL ALLERGY, Issue 6 2007J. Sun Summary Background Seasonal rhinitis is manifested by a series of nasal symptoms in response to exposure to seasonal allergens including ragweed pollen. Understanding its immunological mechanisms may help to better manage the disease. Objective We sought to determine comprehensively ragweed-induced cytokine and chemokine production by peripheral blood mononuclear cells from normal individuals and patients with seasonal rhinitis sensitized to ragweed pollen, and to assess its regulation by exogenous IL-10. Methods Cells were cultured in the presence or absence of a purified ragweed pollen extract with or without exogenous IL-10. Cytokines and chemokines were measured in the supernatant. Gene expression was evaluated using real-time quantitative reverse transcription PCR. Results Ragweed stimulation significantly increased the production of the Th2-associated cytokines IL-5, IL-9 and IL-13, the chemokines CCL17 and CCL22 and the regulatory cytokine IL-10 in allergic patients, whereas transforming growth factor-, (TGF-,) production was increased only in normal individuals. No difference was detected between groups in the production of the Th1 cytokine IFN-, or the Th1-affiliated chemokines CXCL10 and CXCL11. Exogenous IL-10 significantly suppressed spontaneous and induced production of both Th1- and Th2-associated cytokines and chemokines. Conclusion Our work demonstrated that locally manifested allergic rhinitis is underlined by a systemic Th2 immune response specific to allergens. The molecular pathogenesis of allergic rhinitis may be linked to a compromised allergen-specific immune regulation, e.g., reduced spontaneous and allergen-induced TGF-, production in patients compared with healthy controls. Our data also show that IL-10 inhibits both the effector and directional mechanisms of allergen-specific immune response, further supporting its potential therapeutic benefit in preventing and treating allergic diseases. [source] | |||||||||||||