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RNA Analysis (rna + analysis)
Selected AbstractsTargeted gene analysis in Ulmus americana and U. pumila tissuesFOREST PATHOLOGY, Issue 2 2008C. Nasmith Summary Steady-state gene expression was compared between Dutch elm disease (DED)-susceptible Ulmus americana and DED-resistant U. pumila callus, leaf midrib, root and inner bark tissues. Stress-related cDNAs including phenylalanine ammonia-lyase (PAL), chitinase (CHT) and polygalacturonase-inhibiting protein (PGIP) were isolated and compared following RT-PCR of elm tissues. Complete CHT and partial PAL and PGIP cDNA transcripts were identified, each displaying sequence variation between elm species. These transcripts were Dig-labelled and subsequently used for northern analyses of the elm tissues. Midrib and root tissue displayed highest steady-state gene expression compared with inner bark and callus tissues. A modified nucleic acid isolation technique was necessary for downstream RNA analyses. Lithium chloride and polyvinylpyrrolidone were critical for efficient removal of polysaccharides and phenolics associated with some of the elm tissues. Steady-state gene expression is discussed in relation to the tissues investigated. The use of tissues other than in vitro callus culture more closely represents the tissues associated with the elm's vascular response to DED. [source] Genotype,phenotype correlations in hereditary familial retinoblastoma,HUMAN MUTATION, Issue 3 2007Melissa Taylor Abstract We studied 50 unrelated pedigrees with a family history of retinoblastoma (Rb) (165 carriers of a RB1 mutation) to delineate the spectrum of RB1 germline mutations in familial Rb and to identify genotype,phenotype correlations as well as putative modifiers. Patients were followed at Institut Curie and they were examined by an ophthalmologist, a pediatrician, and a geneticist. All cases of familial Rb were determined via genetic counseling. Clinical features included disease status, laterality, age at diagnosis, mutation type, follow-up, and disease,eye ratio (DER). To eliminate mosaic cases, first-generation carriers displaying low-penetrance (LP) Rb were excluded from the analysis. Complete penetrance was the rule for nonsense and frameshift mutations (25 families) and high penetrance was observed for large rearrangements (eight families). Promoter (two families) and missense (two families) mutations displayed heterogeneous phenotypes and LP. Variable penetrance was observed for splice abnormalities (13 families) and was explained by in/out of frame mutations or respect of functional domains. Surprisingly, two families with the LP g.45867G>T/IVS6+1G>T mutation presented data that conflicted with the data reported in previous publications, as unaffected carriers had paternally inherited mutant alleles. Moreover, RNA analyses suggested that the lack of penetrance in unaffected carriers could be explained by an increase in expression levels of the wild-type allele. This observation prompted us to define a new class "3" of LP alleles. We believe this is the first large-scale study of familial Rb with a high level of homogeneity in the clinical and genetic analysis of patients and their relatives, thereby allowing for reliable intrafamilial genotype,phenotype correlations. Our analysis suggests in some cases the influence of modifier factors probably involved in mRNA level regulation and/or pRB pathway regulation. Hum Mutat 28(3), 284,293, 2007. © 2006 Wiley-Liss, Inc. [source] SigB, an RNA polymerase sigma factor required for osmoprotection and proper differentiation of Streptomyces coelicolorMOLECULAR MICROBIOLOGY, Issue 1 2001You-Hee Cho A gene (sigB) encoding an alternative sigma factor ,B in Streptomyces coelicolor A3(2) was isolated and characterized. It encodes a polypeptide of 281 amino acids (31 546 Da) and is highly homologous to Bacillus subtilis,B. The sigB coding region is preceded by four open reading frames (ORFs): dpsA, orfA, rsbB and rsbA in sequential order. RNA analyses revealed that rsbB, rsbA and sigB constitute an operon (sigB operon). Transcripts were produced constitutively from a promoter (sigBp2) upstream of the rsbB coding region, contributing to the basal level expression of ,B protein. An inducible promoter (sigBp1) resembling the catB promoter (catBp) was located between the rsbA and sigB coding regions. Transcripts from sigBp1 dramatically increased as cells differentiated on solid media, at the stationary phase in liquid media or by osmotic stresses similar to the behaviour of catBp transcripts. Both catBp and sigBp1 promoters were recognized specifically by ,B -containing RNA polymerase in vitro. Disruption of the sigB gene abolished not only the differentiation-associated expression but also the osmotic induction of the catB gene, indicating that catBp is under the control of ,B. The sigB mutant exhibited a similar phenotype to the catB mutant, being sensitive to hyperosmolarity, blocked in forming aerial mycelium and with skewed antibiotic production. Therefore, we conclude that ,B ensures the proper differentiation and osmoprotection of S. coelicolor cells, primarily via regulation of the expression of catalase B. [source] Remodeling of extracellular matrix at ovulation of the bovine ovarian follicleMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 10 2006H.F. Irving-Rodgers Abstract Using immunohistochemistry and RNA analyses we examined the fate of components of a newly identified matrix that develops between granulosa cells (focimatrix, abbreviated from focal intraepithelial matrix) and of the follicular basal lamina in ovulating bovine ovarian follicles. Pre- and postovulatory follicles were generated by treatment with estradiol (Day 1), progesterone (Days 1,10), and prostaglandin analogue (Day 9) with either no further treatment (Group 1, n,=,6) and or with 25 mg porcine LH (Day 11, Group 2, n,=,8 or Day 10, Group 3, n,=,8) and ovariectomy on Day 12 (12,14 hr post LH in Group 2, 38,40.5 hr in Group 3). In the time frame examined no loss of follicular basal lamina laminin chains ,2 and ,1 or nidogen 1 was observed. In the follicular basal lamina collagen type IV ,1 and perlecan were present prior to ovulation; after ovulation collagen type IV ,1 was discontinuously distributed and perlecan was absent. Versican in the theca interna adjacent to the follicular basal lamina in preovulatory follicles was not observed post ovulation, however, the granulosa cells then showed strong cytoplasmic staining for versican. Expression of versican isoforms V0, V1, and V3 was detected at all stages. Focimatrix was observed in preovulatory follicles. It contained collagen type IV ,1, laminins ,2 and ,1, nidogen 1 and perlecan and underwent changes in composition similar to that of the follicular basal lamina. In conclusion focimatrix and the follicular basal lamina are degraded at ovulation. Individual components are lost at different times. Mol. Reprod. Dev. © 2006 Wiley-Liss, Inc. [source] A Review of Molecular Contrasts Between Arresting and Viable Porcine Attachment SitesAMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY, Issue 6 2007Jocelyn M. Wessels Significant spontaneous fetal loss of unknown cause occurs in North American commercial swine. About 30% of conceptuses, thought to be genetically normal, are lost during the peri-attachment period. An additional 20% are lost at mid-pregnancy. Littermate endometrial and trophoblast biopsies were studied by quantitative real-time PCR for gene expression, and immunohistochemistry for protein expression at gestation day (gd)15,23 and 50. RNA analyses were also conducted on endometrial lymphocytes and arterial endothelial cells removed from biopsies by laser capture microdissection. Genes were selected for study from human literature and cloned as required. As in humans, angiogenic, cytokine, chemokine and chemokine decoy receptor gene expression occurs at the porcine maternal,fetal interface. In each tissue studied, distinct patterns of expression are found between early and mid-pregnancy, as well as between viable and arresting conceptus attachment sites. These changes involve both endometrial lymphocytes and dendritic cells. Restriction in endometrial angiogenesis, reduction in expression of the chemokine decoy receptor D6, and reduction in dendritic cell numbers contribute to fetal arrest. In peri-attachment loss, interferon-, is more abundantly transcribed than tumor necrosis factor-,, but this ratio is reversed during midgestation failure. Further characterization of spontaneous fetal loss in pigs will identify targets for modification by hog producers and may provide a model for identification of antecedents to fetal loss in humans. [source] Intronic variants in BRCA1 and BRCA2 that affect RNA splicing can be reliably selected by splice-site prediction programs,HUMAN MUTATION, Issue 1 2009Maaike P.G. Vreeswijk Abstract A large number of sequence variants identified in BRCA1 and BRCA2 cannot be distinguished as either disease-causing mutations or neutral variants. These so-called unclassified variants (UVs) include variants that are located in the intronic sequences of BRCA1 and BRCA2. The purpose of this study was to assess the use of splice-site prediction programs (SSPPs) to select intronic variants in BRCA1 and BRCA2 that are likely to affect RNA splicing. We performed in vitro molecular characterization of RNA of six intronic variants in BRCA1 and BRCA2. In four cases (BRCA1, c.81,6T>A and c.4986+5G>T; BRCA2, c.7617+2T>G and c.8754+5G>A) a deleterious effect on RNA splicing was seen, whereas the c.135-15_-12del variant in BRCA1 showed no effect on RNA splicing. In the case of the BRCA2 c.68,7T>A variant, RNA analysis was not sufficient to establish the clinical significance. Six SSPPs were used to predict whether an effect on RNA splicing was expected for these six variants as well as for 23 intronic variants in BRCA1 for which the effect on RNA splicing has been published. Out of a total of 174 predictions, 161 (93%) were informative (i.e., the wild-type splice-site was recognized). No false-negative predictions were observed; an effect on RNA splicing was always predicted by these programs. In four cases (2.5%) a false-positive prediction was observed. For DNA diagnostic laboratories, these programs are therefore very useful to select intronic variants that are likely to affect RNA splicing for further analysis. Hum Mutat 0,1,8, 2008. © 2008 Wiley-Liss, Inc. [source] Evaluation of in silico splice tools for decision-making in molecular diagnosis,HUMAN MUTATION, Issue 7 2008Claude Houdayer Abstract It appears that all types of genomic nucleotide variations can be deleterious by affecting normal pre-mRNA splicing via disruption/creation of splice site consensus sequences. As it is neither pertinent nor realistic to perform functional testing for all of these variants, it is important to identify those that could lead to a splice defect in order to restrict transcript analyses to the most appropriate cases. Web-based tools designed to provide such predictions are available. We evaluated the performance of six of these tools (Splice Site Prediction by Neural Network [NNSplice], Splice-Site Finder [SSF], MaxEntScan [MES], Automated Splice-Site Analyses [ASSA], Exonic Splicing Enhancer [ESE] Finder, and Relative Enhancer and Silencer Classification by Unanimous Enrichment [RESCUE]-ESE) using 39 unrelated retinoblastoma patients carrying different RB1 variants (31 intronic and eight exonic). These 39 patients were screened for abnormal splicing using puromycin-treated cell lines and the results were compared to the predictions. As expected, 17 variants impacting canonical AG/GT splice sites were correctly predicted as deleterious. A total of 22 variations occurring at loosely defined positions (±60 nucleotides from an AG/GT site) led to a splice defect in 19 cases and 16 of them were classified as deleterious by at least one tool (84% sensitivity). In other words, three variants escaped in silico detection and the remaining three were correctly predicted as neutral. Overall our results suggest that a combination of complementary in silico tools is necessary to guide molecular geneticists (balance between the time and cost required by RNA analysis and the risk of missing a deleterious mutation) because the weaknesses of one in silico tool may be overcome by the results of another tool. Hum Mutat 29(7), 975,982, 2008. © 2008 Wiley-Liss, Inc. [source] Failure of the ammonia oxidation process in two pharmaceutical wastewater treatment plants is linked to shifts in the bacterial communitiesJOURNAL OF APPLIED MICROBIOLOGY, Issue 5 2005L. Wittebolle Abstract Aims:, To investigate whether two different wastewater treatment plants (WWTPs) , treating the same pharmaceutical influent , select for a different bacterial and/or ammonia oxidizing bacterial (AOB) community. Methods and Results:, Molecular fingerprinting demonstrated that each WWTP had its own total bacterial and AOB community structure, but Nitrosomonas eutropha and N. europea were dominant in both WWTP A and B. The DNA and RNA analysis of the AOB communities revealed different patterns; so the most abundant species may not necessarily be the most active ones. Nitritation failures, monitored by chemical parameter analysis, were reflected as AOB community shifts and visualized by denaturing gradient gel electrophoresis (DGGE)-based moving window analysis. Conclusions:, This research demonstrated the link between functional performance (nitritation parameters) and the presence and activity of a specific microbial ecology (AOB). Clustering and moving window analysis based on DGGE showed to be valuable to monitor community shifts in both WWTPs. Significance and Impact of the Study:, This study of specific community shifts together with functional parameter analysis has potential as a tool for relating functional instability (such as operational failures) to specific-bacterial community shifts. [source] Identification of Cephalopod Species (Ommastrephidae and Loliginidae) in Seafood Products by Forensically Informative Nucleotide Sequencing (FINS)JOURNAL OF FOOD SCIENCE, Issue 5 2002M.J. Chapela ABSTRACT: An identification technique of commercially important cephalopods based on 16s RNA analysis was developed. A set of primers was designed to amplify a fragment of approximately 200 bp that presents enough variability for reliable species identification. Sequences from this fragment of 9 different authentic species were studied, genetic distances were measured, and a phylogenetic tree was constructed, with individuals of the same species grouped within the same cluster. Eight different types of commercial seafood products, mainly labelled as "squid rings", were analyzed and sequences were employed for species identification showing that FINS is a suitable technique for identification of processed cephalopods. [source] Biological and Molecular Characterization of Taiwanese Isolates of Cucumber mosaic virus Associated with Banana Mosaic DiseaseJOURNAL OF PHYTOPATHOLOGY, Issue 2 2009Chun-Nan Chou Abstract Banana mosaic disease (BMD) caused by Cucumber mosaic virus (CMV) has become an important threat to the banana industry. We collected and characterized 10 CMV isolates associated with BMD in Taiwan and compared their biological characteristics and coat protein sequences. The isolates fell into four pathotypes on the basis of the symptoms they induce on banana, Nicotiana glutinosa and Vigna unguiculata (cowpea). Double-stranded RNA analysis revealed that the different pathotypes are not related to the presence of CMV satellite RNA. Phylogenetic analysis of worldwide CMV coat protein sequences revealed that among the currently known CMV subgroups IA, IB and II, subgroup IB is phylogenetically unresolved. Our CMV isolates form a new subgroup, IT, within subgroup I. In addition, we resolved another new CMV subgroup, IS, within subgroup I. The analysis also revealed that isolates within different subgroups can infect the banana. [source] Analysis of mRNA in hemophilia A patients with undetectable mutations reveals normal splicing in the factor VIII geneJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 2 2005O. EL-MAARRI Summary.,Background: haemophilia A (HA) is characterized by partial or total deficiency of factor VIII (FVIII) protein activity. It is caused by a broad spectrum of mutations in the FVIII gene. Despite tremendous improvements in mutation screening methods, in about 2% of HA patients no DNA change could be found, even after sequencing the whole coding part of the FVIII gene including the flanking splice sites, as well as the promotor and the 3, UTR regions. Objectives, patients and methods: In the present study we performed a detailed RNA analysis of three groups of patients. The first included control patients with known splicing defects, the second included two patients with already identified nucleotide changes close to splicing sites, that could potentially alter the normal splicing process, and a third group of 11 unrelated patients whose genomic DNA have already been screened for mutations by DHPLC and direct sequencing with no mutation being identified. Results: Both candidate splice site mutations were shown to result in either skipping or alternative splicing of at least one exon, therefore these DNA changes must be considered as causal for the patients' HA phenotype. In contrast, no abnormalities on the RNA level were observed in any of 11 unrelated patients without mutations in the FVIII gene. Conclusions: These findings exclude mutations that could be located deep in the introns and affecting either normal splicing or lead to mechanisms causing some unknown rearrangements of the FVIII gene. In fact, our results point to the presence of still unknown factor(s) causing HA, which might be either allelic or in the close proximity of the FVIII gene or non-allelic associated with other genetic loci that are involved in the processing of the FVIII protein. [source] The role of the gut flora in health and disease, and its modification as therapyALIMENTARY PHARMACOLOGY & THERAPEUTICS, Issue 8 2002A. L. Hart Summary The gut flora is a vast interior ecosystem whose nature is only beginning to be unravelled, due to the emergence of sophisticated molecular tools. Techniques such as 16S ribosomal RNA analysis, polymerase chain reaction amplification and the use of DNA microarrays now facilitate rapid identification and characterization of species resistant to conventional culture and possibly unknown species. Life-long cross-talk between the host and the gut flora determines whether health is maintained or disease intervenes. An understanding of these bacteria,bacteria and bacteria,host immune and epithelial cell interactions is likely to lead to a greater insight into disease pathogenesis. Studies of single organism,epithelial interactions have revealed the large range of metabolic processes that gut bacteria may influence. In inflammatory bowel diseases, bacteria drive the inflammatory process, and genetic predisposition to disease identified to date, such as the recently described NOD2/CARD15 gene variants, may relate to altered bacterial recognition. Extra-intestinal disorders, such as atopy and arthritis, may also have an altered gut milieu as their basis. Clinical evidence is emerging that the modification of this internal environment, using either antibiotics or probiotic bacteria, is beneficial in preventing and treating disease. This natural and apparently safe approach holds great appeal. [source] Rational development of a HIV-1 gene therapy vectorTHE JOURNAL OF GENE MEDICINE, Issue 10 2003D. S. Anson Abstract Background HIV-1 provides an attractive option as the basis for gene transfer vectors due to its ability to stably transduce non-cycling cell populations. In order to fully utilise the promise of HIV-1 as a vector it is important that the effects of viral cis sequence elements on vector function are carefully delineated. Methods In this study we have systematically evaluated the effect of various cis elements from the HIV-1 YU-2 genome that have been implicated as either affecting vector performance, or HIV-1 replication, on the efficiency of vector production (titre and infectivity). As a measure of the relative safety of vectors their propensity to inadvertently transfer the gagpol gene to transduced cells was assessed. Results Sequences that were found to increase vector titre were from the 5, end of the gag gene, from the 5, and 3, ends of the env gene, from immediately upstream of the polypurine tract, and the central polypurine tract. The substitution of the HIV-1 RRE with heterologous RNA transport elements, or the deletion of the RRE, resulted in greatly reduced vector titres. RNA analysis suggested that the role of the Rev/RRE system extends beyond simply acting as an RNA nuclear export signal. The relative safety of different vector designs was compared and an optimal construct selected. Conclusions Based on our results we have constructed a vector that is both more efficient, and has better safety characteristics, than the widely used pHR, HIV-1 vector construct. Copyright © 2003 John Wiley & Sons, Ltd. [source] Immunohistochemistry and Reverse Transcriptase,Polymerase Chain Reaction as Methods for Diagnostic Determination of Usher Syndrome Type IIa,THE LARYNGOSCOPE, Issue 7 2004Edward Cohn Abstract Objectives/Hypothesis: Patients having null mutations in the USH2A gene do not produce usherin and therefore are not positive for immunohistochemical staining of the usherin protein. Thus, immunostaining for usherin can serve as a reliable diagnostic tool for Usher syndrome type IIa. Study Design: Prospective. Methods: Immunohistochemical staining for usherin was carried out in basement membrane of minor salivary gland tissue from subjects with confirmed Usher syndrome type IIa and from archival minor salivary gland tissue from patients without Usher syndrome as control samples. Quantitative usherin messenger RNA analysis was performed using minor salivary gland biopsy tissue. Results: Five subjects with Usher syndrome type IIa had no immunostaining in minor salivary gland tissue, whereas control minor salivary gland tissue did stain with usherin antibody. No usherin RNA was detected in biopsy specimens from patients with confirmed Usher syndrome IIa. Conclusion: The feasibility was confirmed of diagnosing Usher syndrome type IIa using purified usherin antibody in subjects having two null USH2A mutations. [source] CFTR Rearrangements in Spanish Cystic Fibrosis Patients: First New Duplication (35kb) Characterised in the Mediterranean CountriesANNALS OF HUMAN GENETICS, Issue 5 2010Marķa D. Ramos Summary Developments in quantitative PCR technologies have greatly improved our ability to detect large genome rearrangements. In particular oligonucleotide-based array comparative genomic hybridisation has become a useful tool for appropriate and rapid detection of breakpoints. In this work, we have analysed 80 samples (42 unknown CF alleles) applying three quantitative technologies (MLPA, qPCR and array-CGH) to detect recurrent as well as novel large rearrangements in the Spanish CF population. Three deletions and one duplication have been identified in five alleles (12%). Interestingly, we provide the comprehensive characterisation of the first duplication in our CF cohort. The new CFTRdupProm-3 mutation spans 35.7 kb involving the 5,-end of the CFTR gene. Additionally, the RNA analysis has revealed a cryptic sequence with a premature termination codon leading to a disrupted protein. This duplication has been identified in five unrelated families from Spain, France and Italy with all patients showing the same associated haplotype, which is further evidence for its likely common Mediterranean origin. Overall, considering this and other previous studies, CFTR rearrangements account for 1.3% of the Spanish CF alleles. [source] Decreased expression of heparin-binding epidermal growth factor,like growth factor as a newly identified pathogenic mechanism of antiphospholipid-mediated defective placentationARTHRITIS & RHEUMATISM, Issue 5 2010N. Di Simone Objective Heparin-binding epidermal growth factor,like growth factor (HB-EGF) plays a role in blastocyst implantation and is down-regulated in preeclampsia and in hypertensive pregnancy disorders associated with defective extravillous trophoblast invasion. Defective placentation and severe preeclampsia are also features of the antiphospholipid syndrome (APS). The purpose of this study was to investigate whether abnormal HB-EGF expression plays a pathogenic role in antiphospholipid antibody (aPL),mediated defective placentation. Methods HB-EGF expression in placental tissue was evaluated by Western blotting and messenger RNA analysis in normal and APS placentae. Polyclonal IgG fractions or monoclonal ,2 -glycoprotein I,dependent aPL and their respective controls were investigated for the following 4 features: their binding to human trophoblast monolayers, as determined by cell enzyme-linked immunosorbent assay (ELISA); their effect on HB-EGF expression by Western blotting in trophoblast cell extracts as well as by ELISA as a protein secreted in the culture supernatants; their inhibitory effect on in vitro trophoblast invasiveness, as evaluated by Matrigel assay; and their inhibitory effect on matrix metalloproteinase (MMP) levels, as measured by gelatin zymography. Experiments were also performed in the presence of serial concentrations of heparin or recombinant HB-EGF. Results Placental APS tissue displayed reduced expression of HB-EGF. Polyclonal and monoclonal aPL bound to trophoblast monolayers and significantly reduced the in vitro synthesis and secretion of HB-EGF. Heparin inhibited aPL binding and restored HB-EGF expression in a dose-dependent manner. Addition of recombinant HB-EGF reduced the in vitro aPL-induced inhibition of Matrigel invasiveness as well as MMP-2 levels. Conclusion These preliminary findings suggest that the reduction of aPL-mediated HB-EGF represents an additional mechanism that is responsible for the defective placentation associated with APS and that heparin protects from aPL-induced damage by inhibiting antibody binding. [source] Selective expression of connective tissue growth factor in fibroblasts in vivo promotes systemic tissue fibrosisARTHRITIS & RHEUMATISM, Issue 5 2010Sonali Sonnylal Objective Connective tissue growth factor (CTGF) is a cysteine-rich secreted matricellular protein involved in wound healing and tissue repair. Enhanced and prolonged expression of CTGF has been associated with tissue fibrosis in humans. However, questions remain as to whether CTGF expression alone is sufficient to drive fibrosis. This study was undertaken to investigate whether CTGF alone is sufficient to cause fibrosis in intact animals and whether its effects are mediated through activation of transforming growth factor , (TGF,) signaling or through distinct signal transduction pathways. Methods We generated mice overexpressing CTGF in fibroblasts under the control of the fibroblast-specific collagen ,2(I) promoter enhancer. Tissues such as skin, lung, and kidney were harvested for histologic analysis. Mouse embryonic fibroblasts were prepared from embryos (14.5 days postcoitum) for biochemical analysis. Results Mice overexpressing CTGF in fibroblasts were susceptible to accelerated tissue fibrosis affecting the skin, lung, kidney, and vasculature, most notably the small arteries. We identified a marked expansion of the myofibroblast cell population in the dermis. RNA analysis of transgenic dermal fibroblasts revealed elevated expression of key matrix genes, consistent with a fibrogenic response. CTGF induced phosphorylation of p38, ERK-1/2, JNK, and Akt, but not Smad3, in transgenic mouse fibroblasts compared with wild-type mouse fibroblasts. Transfection experiments showed significantly increased basal activity of the CTGF and serum response element promoters, and enhanced induction of the CTGF promoter in the presence of TGF,. Conclusion These results demonstrate that selective expression of CTGF in fibroblasts alone causes tissue fibrosis in vivo through specific signaling pathways, integrating cues from the extracellular matrix into signal transduction pathways to orchestrate pivotal biologic responses relevant to tissue repair and fibrosis. [source] Ultrahighly Sensitive Homogeneous Detection of DNA and MicroRNA by Using Single-Silver-Nanoparticle CountingCHEMISTRY - A EUROPEAN JOURNAL, Issue 3 2010Fagong Xu Dr. Abstract DNA and RNA analysis is of high importance for clinical diagnoses, forensic analysis, and basic studies in the biological and biomedical fields. In this paper, we report the ultrahighly sensitive homogeneous detection of DNA and microRNA by using a novel single-silver-nanoparticle counting (SSNPC) technique. The principle of SSNPC is based on the photon-burst counting of single silver nanoparticles (Ag NPs) in a highly focused laser beam (about 0.5,fL detection volume) due to Brownian motion and the strong resonance Rayleigh scattering of single Ag NPs. We first investigated the performance of the SSNPC system and then developed an ultrasensitive homogeneous detection method for DNA and microRNA based on this single-nanoparticle technique. Sandwich nucleic acid hybridization models were utilized in the assays. In the hybridization process, when two Ag-NP,oligonucleotide conjugates were mixed in a sample containing DNA (or microRNA) targets, the binding of the targets caused the Ag NPs to form dimers (or oligomers), which led to a reduction in the photon-burst counts. The SSNPC method was used to measure the change in the photon-burst counts. The relationship between the change of the photon-burst counts and the target concentration showed a good linearity. This method was used for the assay of sequence-specific DNA fragments and microRNAs. The detection limits were at about the 1,fM level, which is 2,5 orders of magnitude more sensitive than current homogeneous methods. [source] | ||||||||||||||||