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RNA Extraction (rna + extraction)
Selected AbstractsGene expression signatures in chronic and aggressive periodontitis: a pilot studyEUROPEAN JOURNAL OF ORAL SCIENCES, Issue 3 2004Panos N. Papapanou This pilot study examined gene expression signatures in pathological gingival tissues of subjects with chronic or aggressive periodontitis, and explored whether new subclasses of periodontitis can be identified based on gene expression profiles. A total of 14 patients, seven with chronic and seven with aggressive periodontitis, were examined with respect to clinical periodontal status, composition of subgingival bacterial plaque assessed by checkerboard hybridizations, and levels of serum IgG antibodies to periodontal bacteria assayed by checkerboard immunoblotting. In addition, at least two pathological pockets/patient were biopsied, processed for RNA extraction, amplification and labeling, and used to study gene expression using Affymetrix U-133 A arrays. Based on a total of 35 microarrays, no significantly different gene expression profiles appeared to emerge between chronic and aggressive periodontitis. However, a de novo grouping of the 14 subjects into two fairly robust clusters was possible based on similarities in gene expression. These two groups had similar clinical periodontal status and subgingival bacterial profiles, but differed significantly with respect to serum IgG levels against the important periodontal pathogens Porphyromonas gingivalis, Tannerella forsythensis and Campylobacter rectus. These early data point to the usefulness of gene expression profiling techniques in the identification of subclasses of periodontitis with common pathobiology. [source] CD8+ T-cell interaction with HCV replicon cells: Evidence for both cytokine- and cell-mediated antiviral activityHEPATOLOGY, Issue 6 2003Chen Liu The interaction between the host immune response and infected hepatocytes plays a central role in the pathogenesis of hepatitis C virus (HCV). The lack of a suitable animal or in vitro model has hindered our understanding of the host T-cell/HCV interaction. Our aim was to develop an in vitro model to study the mechanisms of HCV-specific T-cell-mediated antiviral and cytolytic function. The HCV replicon was HLA typed and lymphocytes were obtained from an HLA class I-matched subject. CD8+ T cells were expanded with 2 HCV-specific/HLA-restricted peptides for NS3. Lymphocyte preparations were cocultured with HCV replicon (FCA1) and control (Huh7) cells labeled with 51Cr. After a 48-hour incubation, the cells were harvested for RNA extraction. Standard blocking assays were performed in the presence of anti-interferon gamma (IFN-,), anti-tumor necrosis factor , (TNF-,), and anti-FasL. Cytolytic activity was measured by 51Cr release. HCV replicon cells express homozygous HLA-A11 alleles and present HCV nonstructural proteins. HCV-specific expansion of CD8+ cells led to a 10-fold decrease in HCV replication by Northern blot analysis and 21% specific lysis of FCA1 cells (compared with 2% of control Huh7 cells). Twenty percent of this antiviral activity was independent of T-cell binding, suggesting cytokine-mediated antiviral activity. The CD8+ antiviral effect was markedly reduced by blocking either IFN-, or FasL but was unaffected by blocking TNF-,. In conclusion, HCV-specific CD8+ cells inhibit viral RNA replication by cytokine-mediated and direct cytolytic effects. This T-cell/HCV subgenomic replicon system represents a model for the investigation of CD8 cell interaction with HCV-infected hepatocytes. [source] cDNA-arrays and real-time quantitative PCR techniques in the investigation of chronic achilles tendinosisJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 6 2003Håkan Alfredson The aetiology and pathogenesis of chronic painful Achilles tendinosis are unknown. This investigation aimed to use cDNA arrays and real-time quantitative polymerase chain reaction (real-time PCR) technique to study tendinosis and control tissue samples. Five patients (females mean age 57.1 ± 4.3 (years ±SD)) with chronic painful Achilles tendinosis were included. From all patients, one biopsy was taken from the area with tendinosis and one from a clinically normal area (control) of the tendon. The tissue samples were immediately immersed in RNAlater and frozen at ,80°C until RNA extraction. Portions of pooled RNA from control and tendinosis sites, respectively, were transcribed to cDNA, radioactively labelled (32P), hybridized to cDNA expression arrays, and exposed to phosphoimager screens over night. Expressions of specific genes, shown to be regulated in the cDNA array analysis, were analyzed in the individual samples using real-time PCR. cDNA arrays showed that gene expressions for matrix-metalloproteinase-2 (MMP-2), fibronectin subunit B (FNRB), vascular endothelial growth factor (VEGF), and mitogen-activated protein kinase p38 (MAPKp38) were up-regulated, while matrix-metalloproteinase-3 (MMP-3) and decorin were down-regulated, in tendinosis tissue compared with control tissue. Using real-time PCR, , and , patients showed up-regulation of MMP-2 and FNRB mRNA, respectively. For decorin, VEGF, and MAPKp38, real-time PCR revealed a great variability among patients. Interestingly, the mRNAs for several cytokines and cytokine receptors were not regulated, indicating the absence of an inflammatory process in chronic painful Achilles tendinosis. In conclusion, cDNA-arrays and real-time PCR can be used to study differences in gene expression levels between tendinosis and control tendon tissue. © 2003 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved. [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] The MAGi RNA extraction method: a highly efficient and simple procedure for fresh and dry plant tissuesJOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 1 2009e Gül Ince Abstract BACKGROUND: Samples from different plant species, different organs or tissues at different times of the year, usually show great differences in their cell compositions, pH, and the endogenous RNase activities, decreasing the RNA yield and quality. RESULTS: In this study we describe a reagent and a simple total RNA isolation method for plant organs, tissues and dry seeds. The RNA extraction reagent (MAGi) is non-toxic and can be stored at room temperature for several months to years. The principle of the total RNA extraction is that tissues are lysed in extraction solution with the aid of mortar homogenization,maceration, and cellular proteins, polysaccharides and DNA are removed from the RNA. We tested the reported method on more than 16 different types of plant seed and 15 different tissues and organs of pepper. CONCLUSION: The RNA extraction procedure reported in the present study greatly reduces the time required to isolate dry seed total RNA and other tissues by more than half as compared with the previously reported methods. The range of typical RNA yield and quality represents a significant improvement over existing protocols. The quality is high enough to be considered as suitable method for RT-PCR, cDNA library construction and microarray gene expression studies. Copyright © 2008 Society of Chemical Industry [source] A new and simple method for rapid extraction and isolation of high-quality RNA from grape (Vitis vinifera) berriesJOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 2 2008Francesca Fort Abstract BACKGROUND: A fast and easy method to isolate RNA from grape (Vitis vinifera) berries is proposed. RESULTS: The procedure uses high concentrations of cetyltrimethylammonium bromide (CTAB) and includes boric acid with the purpose of increasing the RNA extraction. Eppendorf tubes can be used since the amount of sample and volumes of reagents needed are small. The method is based on genomic DNA digestion with DNase I, which avoids the disadvantages of using LiCl (e.g. possible amplification of rare transcripts and reduction or even suppression of the activity of RNA-dependent DNA polymerase). The use of DNase reduces the duration of the isolation method to 8 h. The RNA yield (32.9,49.2 µg g,1) and purity indices were satisfactory in all cases. CONCLUSION: Although there is a referenced grape berry method (RNeasy Midi Kit + polyethylene glycol) that is faster than the one proposed, it is 3.7 times more expensive and gives a lower yield. The ability to successfully amplify from the RNA extract was verified by measuring the ,-tubulin and lipoxygenase gene expression. Copyright © 2007 Society of Chemical Industry [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] The incretin hormones GIP and GLP-1 in diabetic rats: effects on insulin secretion and small bowel motilityNEUROGASTROENTEROLOGY & MOTILITY, Issue 3 2009T. Edholm Abstract, Incretin hormones often display inhibitory actions on gut motility. The aim of this study was to investigate if altered responsiveness to glucose-dependent insulinotropic peptide (GIP) and glucagon-like peptide-1 (GLP-1) as regards insulin release and small bowel motility could bring further clarity to the pathophysiology of diabetes in the Goto-Kakizaki (GK) rat. The isolated perfused pancreas was studied in male GK and Wistar rats (controls) under euglycemic and hyperglycemic conditions. Glucose-dependent insulinotropic peptide (10 nmol L,1) or GLP-1 (10 nmol L,1) were added to the medium and perfusate was collected and analysed for insulin. Moreover, GK and Wistar rats were supplied with bipolar electrodes in the small bowel and myoelectric activity was recorded during intravenous administration of GIP (1,400 pmol kg,1 min,1) or GLP-1 (0.1,20 pmol kg,1 min,1). Finally, tissue was collected from GK and Wistar rats for RNA extraction. Under euglycemia, GIP and GLP-1 stimulated the initial insulin response by 10-fold in GK rats (P < 0.05). At later hyperglycemia, the insulin response to GIP and GLP-1 was blunted to about one-third compared with controls (P < 0.05). In the bowel GLP-1 was about 2.6,16.7 times more potent than GIP in abolishing the migrating myoelectric complex in the GK and control rats. Polymerase chain reaction (PCR) showed GIP and GLP-1 receptor gene expression in pancreatic islets and in small bowel. The initially high, but later low insulin responsiveness to stimulation with GIP and GLP-1 along with inhibition of small bowel motility in the GK rat indicates a preserved incretin response on motility in diabetes type 2. [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] Dynamic culture of droplet-confined cell arraysBIOTECHNOLOGY PROGRESS, Issue 1 2010Elisa Cimetta Abstract Responding to the need of creating an accurate and controlled microenvironment surrounding the cell while meeting the requirements for biological processes or pharmacological screening tests, we aimed at designing and developing a microscaled culture system suitable for analyzing the synergic effects of extracellular matrix proteins and soluble environments on cell phenotype in a high-throughput fashion. We produced cell arrays deposing micrometer-scale protein islands on hydrogels using a robotic DNA microarrayer, constrained the culture media in a droplet-like volume and developed a suitable perfusion system. The droplet-confined cell arrays were used either with conventional culture methods (batch operating system) or with automated stable and constant perfusion (steady-state operating system). Mathematical modeling assisted the experimental design and assessed efficient mass transport and proper fluidodynamic regimes. Cells cultured on arrayed islands (500 ,m diameter) maintained the correct phenotype both after static and perfused conditions, confirmed by immunostaining and gene expression analyses through total RNA extraction. The mathematical model, validated using a particle tracking experiment, predicted the constant value of velocities over the cell arrays (less than 10% variation) ensuring the same mass transport regime. BrdU analysis on an average of 96 cell spots for each experimental condition showed uniform expression inside each cell island and low variability in the data (average of 13%). Perfused arrays showed longer doubling times when compared with static cultures. In addition, perfused cultures showed a reduced variability in the collected data, allowing to detect statistically significant differences in cell behavior depending on the spotted ECM protein. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010 [source] Detection of micrometastases in lymph nodes from patients with breast cancerBRITISH JOURNAL OF SURGERY (NOW INCLUDES EUROPEAN JOURNAL OF SURGERY), Issue 1 2002G. Branagan Background: Sentinel node biopsy affords the opportunity of focused examination of lymph nodes, including the use of the reverse transcriptase,polymerase chain reaction (RT-PCR). The mammaglobin gene is expressed by breast cancers but has not been detected in histologically normal lymph nodes. This study compared mammaglobin RT-PCR with routine histology in the sentinel and non-sentinel nodes of patients with breast cancer. Methods: Patients with breast cancer underwent tumour excision, sentinel node biopsy and axillary dissection. All nodes were bisected and half of each node was sent for routine histological examination. The other half underwent RNA extraction and mammaglobin RT-PCR. Results: Sentinel node biopsy was successful in 50 (96 per cent) of 52 patients. Mammaglobin expression was detected in nine (8 per cent) of 119 histologically negative sentinel nodes (Clopper,Pearson 95 per cent confidence interval (c.i.) 4 to 14 per cent) and in 13 (5 per cent) of 247 histologically negative non-sentinel nodes (95 per cent c.i. 3 to 9 per cent). Mammaglobin expression was detected in four (13 per cent) of 31 patients with histologically negative sentinel nodes (95 per cent c.i. 4 to 30 per cent) and in six (14 per cent) of 44 patients with histologically negative non-sentinel nodes (95 per cent c.i. 5 to 27 per cent). The false-negative rate for sentinel node biopsy was zero using histology results and 10 per cent using RT-PCR. Conclusion: RT-PCR screening of axillary nodes for mammaglobin expression increased the detection of breast cancer metastases compared with routine histology. © 2002 British Journal of Surgery Society Ltd [source] Prognostic significance of RET and NTRK1 rearrangements in sporadic papillary thyroid carcinomaBRITISH JOURNAL OF SURGERY (NOW INCLUDES EUROPEAN JOURNAL OF SURGERY), Issue 9 2000T. J. Musholt Background The genetic background of papillary thyroid carcinoma (PTC) has been elucidated by the identification of somatic translocations of the tyrosine kinases RET and NTRK1. Expression of RET/PTC chimeras has been demonstrated in 10,25 per cent of sporadic PTCs while rearrangements of NTRK1 were detected less frequently. Based upon the limited data available, some investigators have hypothesized that RET/PTC activation is preferentially associated with slow growing tumours of low malignancy in elderly patients, while other studies support the contrary. Methods Tumour tissues from 115 patients with sporadic PTC were harvested at operation and snap frozen. Following RNA extraction, expression analysis of the RET proto-oncogene as well as the NTRK1 gene was performed by multiplex reverse transcriptase,polymerase chain reaction. Samples with suspected rearrangements of the genes were further analysed for expression of the hybrid messenger RNAs RET/PTC1 to RET/PTC4, and for known NTRK1 chimeras. Clinical data of all patients were documented in an extensive database of thyroid carcinomas maintained by this research group. Results Twenty-one (18 per cent) of 115 tumour samples revealed somatic rearrangements of RET while translocations of the NTRK1 gene were demonstrated in 2 per cent of all samples analysed to date. The mean age of all patients was 52 (range 14,86, median 54) years and that of patients positive for RET rearrangements was 49 (range 14,82, median 49) years. Nine of 21 RET -rearranged tumours showed aggressive behaviour with locally invasive tumour growth and infiltration of adjacent structures such as muscles, vessels and trachea. Tumour samples without detectable RET translocations were associated with organ-exceeding tumour growth in only 20 per cent of cases. Conclusion These data represent one of the most comprehensive studies on gene translocations and their clinical significance in PTC. In accordance with international publications, an incidence of 2 per cent of NTRK1 rearrangements and 18 per cent of RET translocations is reported, which is in contrast to other national reports of low rearrangement rates. Somatic translocations were associated with tumours demonstrating aggressive behaviour in almost half of patients with PTC in all age groups, indicating a prognostic role of oncogenic RET/PTC activation. © 2000 British Journal of Surgery Society Ltd [source] Differential gene expression analysis using paraffin-embedded tissues after laser microdissectionJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 5 2003Joung-Ok Kim Abstract Recent advances in laser microdissection allow for precise removal of pure cell populations from morphologically preserved tissue sections. However, RNA from paraffin-embedded samples is usually degraded during microdissection. The purpose of this study is to determine the optimal fixative for RNA extractions from laser microdissected paraffin-embedded samples. The integrity of RNA was evaluated with the intactness of 18S and 28S ribosomal RNA by electrophoresis and by the length of individual gene transcripts using RT-PCR. The various fixatives were methacarn (a combination of methanol, chloroform, and acetic acid) and several concentrations of ethanol and isopropanol. Methacarn was the optimal fixative for RNA preservation in paraffin-embedded tissues, which included liver, lung, kidney, muscle, and limb. Based on RT-PCR analysis, methacarn fixed samples exhibited the expected RNA sizes for individual genes such as glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) and bone-related genes (e.g., alkaline phosphatase and osteonectin). The laser microdissection technique with methacarn fixation was then applied to analyze the differential gene expression between hypertrophic and proliferative chondrocytes in the growth plate of long bone. The expression of type X collagen (ColX,1), a specific gene for hypertrophic chondrocytes, was only observed in hypertrophic chondrocytes, while type II collagen (Col2,1) was observed more broadly in the growth plate as anticipated. Thus, combining laser microdissection with methacarn fixation facilitates the examination of differentially expressed genes from various tissues. © 2003 Wiley-Liss, Inc. [source] Evaluation of different RNA extraction methods for small quantities of plant tissue: Combined effects of reagent type and homogenization procedure on RNA quality-integrity and yieldPHYSIOLOGIA PLANTARUM, Issue 1 2006Mary Portillo Highly sensitive techniques for transcriptome analysis, such as microarrays, complementary DNA-amplified fragment length polymorphisms (cDNA-AFLPs), and others currently used in functional genomics require a high RNA quality and integrity, as well as reproducibility among extractions of replicates from the same tissue. There are, however, few technical papers comparing different homogenization techniques and reagents to extract RNA from small quantities of plant tissue. We extracted RNA from tomato seedlings with the three different commercial reagents TRIZOL LS®, TRIZOL®, and TRI Reagent® in combination with pulverization, homogenization-maceration in a mortar, and homogenization with mild vibration plus glass beads, and evaluated total RNA integrity-quality and yield. Pulverization under liquid nitrogen combined with TRIZOL LS® as extraction reagent and homogenization-maceration in mortar with TRI Reagent®, are the procedures that rendered higher RNA yield, integrity and quality, as well as reproducibility among independent RNA extractions. In contrast, short mild vibration pulses (4500 r.p.m. for 5 s) mixed with glass beads, rendered low extraction efficiency and caused, in most cases, partial RNA degradation. [source] | |||||||||||||