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cDNA Synthesis (cdna + synthesis)
Selected AbstractsIdentification of novel genes regulated by ,-melanocyte-stimulating hormone in murine bone marrow-derived dendritic cellsEXPERIMENTAL DERMATOLOGY, Issue 9 2004T. Brzoska Many strains of evidence indicate that ,-melanocyte-stimulating hormone (,-MSH) elicits its immunomodulatory activity via binding to melanocortin receptors (MC-Rs) expressed on monocytes and dendritic cells. In order to identify novel target genes regulated by ,-MSH in these cells, we prepared bone marrow-derived dendritic cell precursors from BALB/c mice and treated them with GM-CSF and IL-4 for 6 days. The MC-R profile on these immature dendritic cells was first determined by quantitative RT-PCR. Both transcripts for MC-1R and MC-5R were detected in these cells. Cells were subsequently stimulated with dinitrobenzene sulfonic acid (DNBS), ,-MSH or both substances for 2 or 16 h. After RNA preparation, cDNA synthesis and in vitro transcripton hybridization of biotinylated cRNA samples was performed on MG U74A Affymetrix gene chips. Data evaluation, cleansing, extraction and analysis of the more than 12 000 cloned genes and expressed sequence tags were performed using the GENE DATA ANALYST vs. 1 Expressionist software. Filter criteria included a minimum threshold of 100, normalization by the logarithmic mean and a quality setting of P < 0.04. Changes with a change factor of >2 were regarded as significant. As expected, stimulation with DNBS resulted in induction or upregulation of genes encoding proinflammatory cytokines, growth factors, signal transduction intermediates and transcription factors. Treatment with ,-MSH blocked the DNBS-driven upregulation of several known genes such as IL-1 or CD86. On the other hand, ,-MSH modulated the expression of several novel genes implicated in immunomodulation, e.g. IL-1, converting enzyme, IFN-, receptor, FK506-binding proteins or several neuropeptides and their receptors. These data indicate novel molecular targets by which ,-MSH exerts its immunomodulatory activities in immunocompetent cells. [source] Highly sensitive detection of the MGB1 transcript (mammaglobin) in the peripheral blood of breast cancer patientsINTERNATIONAL JOURNAL OF CANCER, Issue 4 2004Nuno Cerveira Abstract We describe a new one-step RT-PCR assay for the detection of the mammaglobin (MGB1) gene transcript in the peripheral blood of breast cancer patients. With this approach, the MGB1 transcript could be detected in the peripheral blood of 22 of 54 (41%) breast cancer patients prior to any therapy. This method, using specific primers for cDNA synthesis, proved to be more sensitive (10,6 to 10,11, usually 10,7) than previously reported methodologies. This increased sensitivity was achieved without compromising specificity, as the MGB1 transcript was not detected in 38 blood samples of healthy donors and in only 1 of 18 blood samples of patients presenting with hematologic malignancies. A positive correlation was seen between MGB1 positivity and breast cancer stage: 0/3 (0%) in stage 0, 3/13 (23%) in stage I, 6/17 (35%) in stage II, 5/10 (50%) in stage III, 8/11 (73%) in stage IV (p = 0.003). The prognostic and therapeutic implications of MGB1 positivity by one-step RT-PCR in the peripheral blood of breast cancer patients, especially in clinically localized disease (stages I and II), should be evaluated after long-term clinical follow-up of these patients. © 2003 Wiley-Liss, Inc. [source] HCV-RNA In Sural Nerve From Hcv Infected Patients With Peripheral NeuropathyJOURNAL OF THE PERIPHERAL NERVOUS SYSTEM, Issue 1 2001L De Martino Objective: Evaluation of hepatitis C virus (HCV) by reverse transcription-polymerase chain reaction (RT-PCR) in peripheral nerve tissues from HCV infected patients with peripheral neuropathy. METHODS: RT-PCR was performed on homogenates of nerve biopsies from 17 consecutive HCV-positive patients with peripheral neuropathy, with or without mixed cryoglobulinemia, hospitalised from 1996 to 2000. Sural nerve specimens were frozen in iso-pentane pre-cooled in liquid nitrogen and stored at ,80°C until use. RNA was extracted from ten 7-,m thick cryostatic sections or from a nerve trunk specimen of about 3 mm length, collected from each biopsy. Three different protocols of RNA extraction were tested (1,3). Complementary DNAs (cDNAs) were obtained without or with RNasin (Promega, Madison, WI) addition in the reaction mixture to inhibit residual RNase activity. Two sets of commercially available PCR primers for the outer and the nested reaction were used. PCR products were analysed by agarose gel electrophoresis and ethidium bromide staining. Serum samples and liver specimens from proven HCV positive patients served as positive controls, whereas sera from healthy subjects were negative controls. RESULTS: Sufficient amount of RNA could be obtained either by cryostatic sections or by in toto nerve specimens. Extraction by Trizol (Gibco-BRL) allowed the best concentration and purity of RNA as assessed by biophotometry. The presence of RNasin didn't improve the cDNA synthesis. The resulting amplification product of the nested PCR was 187 bp long. We have always observed this product in our positive controls and never in the negative. Six samples from patients either with or without cryoglobulinemia resulted positive; 7 were negative. Four samples gave variable results. CONCLUSIONS: While 40% of the nerves in our series were undoubtedly HCV positive, the cause(s) of negative and variable results in the remaining samples is likely more complex than variations in the detection protocols and deserve further investigations. REFERENCES: 1) Chomczynski P, Sacchi N (1987). Anal Biochem 162:156. 2) Marquardt O et al. (1996). Med Microbiol Lett 5:55. 3) Chomczynski P (1993). Bio/Techniques 15:532. [source] NSAID-induced antral ulcers are associated with distinct changes in mucosal gene expressionALIMENTARY PHARMACOLOGY & THERAPEUTICS, Issue 1 2009J. C. DESAI Summary Background, The basis for individual variation in gastroduodenal vulnerability to NSAIDs is not well understood. Aim, To assess whether a gene expression signature is associated with susceptibility to gastroduodenal ulcerations. Methods, Twenty-five Helicobacter pylori negative adults were treated for 7 days with naproxen 500 mg b.d. Subjects underwent baseline and post-treatment endoscopy, during which biopsies were taken from antrum and duodenum. RNA extraction and cDNA synthesis were performed, followed by PCR of 23 genes relevant to mucosal injury and repair. Fold changes in gene expression were compared between subjects who developed ulcers and those who did not. Results, Compared with subjects who did not develop ulcers (n = 18), subjects who developed antral ulcers (n = 7) had significantly greater mucosal up-regulation of interleukin-8 [Fold change = 33.5 (S.E.M. = 18.5) vs. ,7.7 (3.2)] and of cyclo-oxygenase-2 [2.3 (1.7) vs. ,10.8 (2.2)]. Conversely, non-ulcer subjects had significantly greater up-regulation of toll-like receptor-4, cyclo-oxygenase-1 and hepatocyte growth factor [14.0 (2.2) vs. ,0.8 (1.0), 9.8 (2.4) vs. 0.0 (0.7) and 8.2 (2.6) vs. ,2.2 (0.3) respectively]. Conclusions, NSAID-induced antral ulcers are associated with a specific pattern of gastroduodenal mucosal gene expression. These patterns may provide an insight into the molecular basis of individual susceptibility to mucosal injury. [source] Identification and characterization of Japanese encephalitis virus envelope protein gene from swineLETTERS IN APPLIED MICROBIOLOGY, Issue 1 2010J.-M. Fan Abstract Aims:, Identification and characterization of Japanese encephalitis virus (JEV) envelope protein gene from swine. Methods and Results:, Genomic RNA was separated from JEV isolated strain Henan-09-03, and used as templates for cDNA synthesis of E gene. The cDNA of E gene was amplified by RT-PCR and cloned into the pMD19-T-Vector and confirmed by sequencing. The cloned gene was then subcloned into the pET-32a and was introduced into Escherichia coli BL21 (DE3) for expression. The E protein was purified by Ni chelating column-based affinity chromatography. The molecular weight of expressed protein was about 50 kDa. Compared with the published sequence of SA14 (AF495589), the homology of the nucleotide sequence was 98% and the seven mutations resulting in amino acid substitutions at Leu 36 Ser, Leu107 Val, Ala167 Thr, Asn 230 Ser, Leu 340 Pro, Asn 430 Ile, Phe 448 Leu. Phylogenetic analysis of the E sequence of isolated strain classified it within genotype III of the JEV. The result of Western blotting indicated that the antigenicity of the protein was specific. Conclusions:, The stable expression of the protein and the analysis of its antigenic specificity provide the foundation for developing the ELISA early stage diagnosis kit. Significance and Impact of the Study:, As coating antigen, the recombinant E protein served a good source in the indirect ELISA method for the detection of JEV antibody. [source] Transcriptional Analysis of Buffalo (Bubalus bubalis) Oocytes During In Vitro Maturation Using Bovine cDNA MicroarrayREPRODUCTION IN DOMESTIC ANIMALS, Issue 1 2010OM Kandil Contents The need for improving in vitro production of buffalo embryos necessitates a better understanding of the molecular mechanisms regulating early development including oocyte maturation. Here, we used bovine cDNA microarray platform to investigate mRNA abundance of buffalo oocytes before and after in vitro maturation. For this, a total of six pools each contains 50 immature or in vitro matured buffalo oocytes were used for mRNA isolation and subsequent cDNA synthesis. The BlueChip bovine cDNA microarray (with approximately 2000 clones) was used to analyse gene expression profiles between immature and matured oocytes. Statistical analysis of microarray data revealed a total of 104 transcripts to be differentially expressed between the two oocyte groups. Among these, transcription factors (ZFP91), M-phase mitotic cell cycle (MPHOSPH9), growth factor (BMP15) and DNA binding (HMGN2) were found to be up-regulated in immature oocytes. Similarly, matured oocytes were found to be enriched with genes involved in cytoskeleton (ACTB), hydrogen ion transporting (ATP6V1C2) and structural constituent of ribosome (RPS27A). Quantitative real-time polymerase chain reaction validated the expression profile of some selected transcripts during array analysis. In conclusion, to our knowledge, this is the first large-scale expression study to identify candidate genes differentially abundant and with potential role during buffalo oocyte maturation. [source] Absence of c- kit gene mutations in gastrointestinal stromal tumours from neurofibromatosis type 1 patientsTHE JOURNAL OF PATHOLOGY, Issue 1 2004Kazuo Kinoshita Abstract Most sporadic gastrointestinal stromal tumours (GISTs) have somatic c- kit gene mutations that are considered to be causal. Neurofibromatosis type 1 (NF1) is caused by mutations of the NF1 gene and NF1 patients have an increased risk of developing GISTs. Since most neoplasms are considered to develop as a result of the combination of several gene mutations, these findings suggest that GISTs from NF1 patients might have somatic c- kit gene mutations and that sporadic GISTs from non-NF1 patients might have somatic NF1 gene mutations. The present study analysed 29 GISTs from seven NF1 patients for c- kit gene mutations and ten sporadic GISTs from ten non-NF1 patients for NF1 mutations. Exons 9, 11, 13, and 17 of the c- kit gene were amplified and directly sequenced after the extraction of genomic DNA from wax-embedded tissues from 26 GISTs from five NF1 patients. The whole coding region of the c- kit cDNA and the whole coding region of the NF1 cDNA were amplified and directly sequenced after RNA extraction and cDNA synthesis in three fresh GIST tissues from two NF1 patients and ten fresh GIST tissues from ten non-NF1 patients. Of the ten sporadic GISTs, eight had heterozygous mutations at exon 11, and one at exon 9, of c- kit. Heterozygous NF1 gene mutations were detected in GISTs from the two NF1 patients from whom fresh tissues were available. None of the 29 GISTs derived from NF1 patients had detectable c- kit gene mutations and none of the ten GISTs derived from non-NF1 patients had detectable NF1 mutations. These results suggest that the pathogenesis of GISTs in NF1 patients is different from that in non-NF1 patients. Copyright © 2004 John Wiley & Sons, Ltd. [source] | ||||||||||