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

(R4b) gPRG: Toward Consensus on Glycan Analysis:Reliable Methods and ReproducibilityJ. Zaia 1 , D. Kolarich 2 , R. Orlando 31Boston University, Boston, MA, United States; 2 Max PlanckInstitute of Colloids and Interfaces, Berlin, Germany; 3 TheUniversity of Georgia Carbohydrate Research Center,Atlanta, GA, United StatesThe field has undertaken over the past few years a series of studies toevaluate methods for glycan analysis. There are several methods thatare widely used at this time for characterization of glycoprotein glycansthat appear to be generally reliable. These include (1) reductiveamination followed by reversed phase chromatography, (2) reductiveamination with matrix assisted laser desorption/ionization (MALDI)mass spectrometry (MS), (3) reductive amination with electrosprayliquid chromatography-MS, and (4) permethylation with tandem MS.The ABRF glycoprotein research group (gPRG) is planning a study inwhich participant laboratories carry out glycan analysis according toprotocols representing best practice in the field. The results will bepresented at the 2012 ABRF meeting. The results will be useful fordemonstrating the reproducibility of glycan analysis using comparableprotocols. They will also highlight the extent to which different methodsbias the glycan analysis results obtained. This workshop will summarizethe history and use of the glycan analysis methods proposed for the2012 study. There will be opportunity for discussion and comment onthese methods.(R5) Joint Session: Nucleic Acids Research Group(NARG) & MicroArray Research Group (MARG)(R5a) Determining miRNA Expression Levels inDegraded RNA Samples Using Real-Time RT-qPCRand Microarray TechnologiesS. Chittur 1 , S. Tighe 2 , J. Holbrook 3 , V. Nadella 4 ,R. Carmical 5 , K. Sol-Church 3 , A.T. Yueng 61State University of New York at Albany, Albany, NY, UnitedStates; 2 University of Vermont, Burlington, VT, United States;3Nemours/A.I. duPont Hospital for Children, Wilmington, DE,United States; 4 Ohio University, Athens, OH, United States;5The University of Texas Medical Branch, Galveston, TX ,United States; 6 Fox Chase Cancer Center, Philadelphia, PA,United StatesThe Nucleic Acid Research Group (NARG) has previously conductedstudies evaluating the impact of RNA integrity and priming strategieson cDNA synthesis and real-time RT-qPCR. The results of last year’s fieldstudy as it relates to degraded RNA will be presented. In continuationof the RNA integrity theme, this year’s study was designed to evaluatethe impact of RNA integrity on the analysis of miRNA expressionusing real-time RT-qPCR. Target section was based on data obtainedby the Microarray Research Group (MARG) and other published datafrom next gen sequencing. These 9 miRNAs represent three groupsof miRNA that are expressed at low, medium or high levels in the FirstChoice human brain reference RNA sample. Two popular RT primingstrategies tested in this study include the Megaplex miRNA TaqManassay (ABI) and the RT2 miRNA qPCR assay (Qiagen/SA Biosciences).The basis for the ABI assay design is a target-specific stem-loopstructure and reverse-transcription primer, while the Qiagen designcombines poly(A) tailing and a universal reverse transcription in onecDNA synthesis reaction. For this study, the human brain referenceRNA was subject to controlled degradation using RNase A to RIN (RNAIntegrity Number) values of 7 (good), 4 (moderately degraded), and2 (severely degraded).These templates were then used to assess bothRT methods. In addition to this real-time RT-qPCR data, the same RNAtemplates were further analyzed using universal poly(A) tailing andhybridization to Affymetrix miRNA GeneChips. This talk will provideinsights into RT priming strategies for miRNA and contrast the qPCRresults obtained using different technologies.(R5b) Microarray Research Group Projects, 2010-11D. Baldwin 1 , N.G. Reyero-Vinas 2 , N. Jafari 31Penn Molecular Profiling Facility, University of Pennsylvania,Philadelphia, PA, United States; 2 Jackson State University,Jackson, MS, United States; 3 Northwestern University,Evanston, IL, United StatesMembers of the MARG will discuss our research projects: Comparisonof microarray and deep sequencing platforms for microRNA profiling,Performance of a synthetic human microRNA reference panel,Participation in the SEQC Sequencing Quality Control consortium, andRNA-Seq profiling of environmental samples exposed to the Gulf oilspill.(R6) Joint Session: Protein Expression ResearchGroup (PERG) & Molecular InteractionsResearch Group (MIRG)(R6a) PERG Research Group Presentation: RefoldingStudyC. KinslandCornell University, Ithaca, NY, United StatesCurrently, the overwhelming majority of protein purification projectsstart with a recombinant protein expressed in a suitable host. For arange of reasons, E. coli is the predominant expression host yet a largepercentage of proteins expressed therein are found in an insolubleform called inclusion bodies. Inclusion bodies have the advantage ofconsisting of relatively homogeneous protein, which can simplify thepurification process. However, this leaves the challenge of solubilizingand refolding the protein into its native and biologically active structure.The conditions for efficient refolding are particular for each protein andthere are a wide range of methods to choose from. For this and otherreasons, many researchers are hesitant to pursue a refolding strategyto obtain a target protein. An overview of refolding methods andstrategies will be presented along with a description of the upcomingProtein Expression Research Group (PERG) refolding study.Research GroupPresentation AbstractsABRF 2011 — Technologies to Enable Personalized Medicine • 57