Conference Program - ABRF 2011 - Association of Biomolecular ...

kits for Tempus TM and PAXgene® stabilized blood. Isolated total RNAwas then processed through the GLOBINclear TM -Human kit and thenamplified with the MessageAmp TM Premier kit creating a library forhybridization to the Affymetrix Human U133A 2.0 expression arrays.Affymetrix GeneChip® microarray analysis showed parameters withinnormal limits for expressed genes. Reported resultsof this controlledexperiment, support a validated gene expression workflow for bloodon Affymetrix expression arrays using a combination of commercial kitsfor RNA purification from stabilized blood and library amplificationoptimized for hybridization and analysis on expression microarrays.159 Performance Comparison of FourMethods Utilized for the Purificationof Enzymatically-Digested,Fluorescently-Labeled PCR FragmentsGenerated During T-RFLP AnalysisM. Zianni, J. Panescu, P. KumarThe Ohio State University, Columbus, OH, UnitedStatesThe fluorescently-labeled Terminal Restriction Fragment LengthPolymorphism (T-RFLP) assay based on amplified ribosomal DNAfrom bacteria is an inexpensive, widely accessible, effective and wellestablishedmolecular technique for the identification and comparativequantification of bacterial species in metagenomics. In order tofacilitate the detection of a large proportion of species in a givensample, it is necessary to maximize the recovery of the fluorescentlylabeled,restriction enzyme-digested PCR fragments generatedduring the process. The post-digestion purification method used tofacilitate fragment separation is a critical step in the retention of DNAfragments. Four methods for post-digestion purification were testedin an effort to characterize their integrity, effectiveness, ease of useand potential biases: solid phase reversible immobilization (AMPure TMand CleanSEQ TM ), contaminant capture (BigDye® XTerminator TM ),and dialysis (Millipore TM Nitrocellulose Membranes). Samples werecollected from four different oral sites in each of 16 patients. Eachsample was divided equally and the DNA was isolated with two distinctmethods. PCR reactions were carried out with two paired universalprimers for the 16S gene that were labeled with VIC and FAM, purifiedwith AMPure TM and digested separately with HhaI and MspI restrictionenzymes. From each of the digestions, identical aliquots were purifiedwith the four different methods. The DNA fragments were analyzedon the Applied BioSystems TM 3730 DNA Analyzer using identicalconditions, followed by data analysis with GeneMapper® v4.0. Basedon preliminary data analysis, CleanSEQ TM is the superior purificationmethod because it resulted in the most numerous peaks recovered persample with a wide distribution of sizes from approximately 50 to 1200bp.160 Electrochemical Simulationof Covalent DNA AdductFormation Monitored with LiquidChromatography/Mass SpectrometryJ. Powers 2 , S. Plattner 1 , R. Erb 1 , F. Pitterl 1 ,J.P. Chervet 2 , H. Oberacher 11Institute of Legal Medicine, Innsbruck MedicalUniversity, Innsbruck, Austria; 2 Antec, Palm Bay, FL,United StatesDNA adduct is a piece of DNA covalently bond to chemicals. Adductsactivate repair processes and, unless repaired prior to replication, maylead to nucleotide substitutions, deletions, and other rearrangements.As alterations of the genetic material can cause severe diseasesincluding cancer, inflammation, and neurodegenerative disorders,tests on mutagenicity/genotoxicity have become an integral part of riskassessment during the development of any new chemical. A number of invitro tests using different cell lines and in vivo tests mainly using rodentsare available to identify hazardous compounds. The majority of thesetests are time-consuming, labor-intensive and hardly automatable. So insearch for alternative methods, the usefulness of electrochemistry (EC)-liquid chromatography (LC)-mass spectrometry (MS) was evaluated.Generally, EC represents a powerful tool to study in vitro biologicaloxidation processes of a number of different compounds, includingdrugs, peptides and proteins. Here, we used EC to initiate adductformation. The obtained reaction products were separated by LC anddetected by MS. Tandem MS experiments were used for structuralconfirmation. In a proof of principle study acetaminophen was selectedas model compound. Covalent adduct formation was observed forelectrochemical activated mixtures of acetaminophen and guanosine.Mechanistic studies revealed that adduct formation will only start atelectrochemical potentials sufficiently high to initiate oxidation of bothacetaminophen and guanosine. The stringent necessity of coactivationsheds a new light on the mechanism of adduct formation, becauseaccording to the generally accepted theory activation of the adductformingagent should be sufficient. Chromatographic separationenabled the differentiation of four isomeric forms. Their connection toacetaminophen was proven in dose-response experiments. EC/LC/MSrepresents a fast, simple, convenient and functional tool to study DNAadduct formation which has great potential to complement the existingbattery of mutagenicity/genotoxicity tests.**161 Quantitative miRNA ExpressionAnalysis Using Fluidigm MicrofluidicsDynamic ArraysJ. Jen, J. Sung Jang, V.A. Simon, R.M. Feddersen,F. Rakhshan, D.A. Schultz, M.A. Zschunke,W.L. Lingle, C.P. Kolbert.Mayo Clinic Micorarray Shared Resource andBiospecimens Accessioning Processing SharedResource, Rochester, MN, United StatesPoster AbstractsMicroRNA (miRNA) is a small non-coding RNA that can regulate geneexpression in both plants and animals. Studies showed that miRNAs playa critical role in human cancer by targeting messenger RNAs that arepositive or negative regulators of cell proliferation and apoptosis. Here,we evaluated miRNA expression in formalin fixed, paraffin embedded(FFPE) samples and fresh frozen (FF) samples using a high throughputqPCR-based microfluidic dynamic array technology (Fluidigm). Wecompared the results to hybridization-based microarray platformsABRF 2011 — Technologies to Enable Personalized Medicine • 77