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

165 Benchmarking miRNA ExpressionLevels in Degraded RNA SamplesUsing Real-Time RT-qPCR andMicroarray TechnologiesJ. Holbrook 5 , S.V. Chittur 1 , S. Tighe 2 , V. Nadell 3 ,R. Carmica 4 , K. Sol-Church 5 , A.T. Yeung 61State University of New York at Albany, Albany,NY, United States; 2 University of Vermont,Burlington, VT, United States; 3 Ohio University,Athens, OH, United States; 4 The University ofTexas Medical Branch, Galveston, TX, UnitedStates; 5 Nemours/A.I. duPont Hospital for Children,Wilmington, DE, United States; 6 Fox Chase CancerCenter, Philadelphia, PA, United StatesThe Nucleic Acid Research Group (NARG) has conducted experimentsto determine the impact of RNA integrity and priming strategies oncDNA synthesis and Real-Time RT-qPCR. As a continuation of the RNAintegrity theme, this year’s study was expanded to evaluate the impactof RNA integrity on priming strategies for the analysis of nine miRNAtargets using Real-Time RT-qPCR. The nine targets were selectedbased on data obtained by the Microarray Research Group (MARG)and represent groups of miRNAs that are expressed at low, medium, orhigh levels in the First Choice human brain reference RNA sample. Thetwo RT-qPCR priming strategies tested in this study include the miRNATaqMan assay (Megaplex) of ABI and the RT2 miRNA qPCR assay ofQiagen/SA Biosciences. The basis for the ABI assay design is a targetspecificstem-loop structure and reverse-transcription primer, whilethe Qiagen design combines poly (A) tailing and a universal reversetranscription in one cDNA synthesis reaction. For this study to assessboth RT methods, samples that were used as templates were humanbrain reference RNA that has been subjected to controlled degradationusing RNase A to RIN (RNA Integrity Number) values of 7 (good), 4(moderately degraded), and 2 (severely degraded). In addition tothis Real-Time RT-qPCR data, the same RNA templates were furtheranalyzed using universal poly (A) tailing followed by hybridizationto Affymetrix miRNA GeneChips. We present some insights into RTpriming strategies for miRNA and contrasts qPCR results obtained usingdifferent technologies.168 MicroRNA Analysis Using RNAExtracted from Matched Formalin-Fixed Paraffin-Embedded (FFPE) andFresh Frozen Samples on SOLiD TMSystemK. Lea, J. Gu, E. Zeringer, S. Heater, J. Schageman,C. Mueller, K. BramlettLife Technologies, Carlsbad, CA, United StatesArchived formalin-fixed paraffin-embedded (FFPE) specimensrepresent excellent resources for biomarker discovery, but it has beena major challenge to study gene expression in these samples due tomRNA degradation and modification during fixation and processing.MicroRNAs (miRNAs) regulate gene expression at post-transcriptionallevel and are considered as important regulators of cancer progression.Next generation sequencing technologies such as SOLiD TM providean ideal method for measuring the abundance of miRNA moleculesin different cancer stages and provide insightful information ontumorigenesis. However, currently there is no good method tosystematically study miRNA expression in FFPE samples on nextgeneration sequencing platforms. We have designed and developeda ligation-based miRNA detection method to capture small RNAsequences in FFPE samples and convert them into templates suitablefor sequencing on the SOLiD TM System. Total RNA was isolated frommatched lung adenocarcinoma FFPE and snap frozen tissues using anAmbion RecoverAll TM kit. A PureLink TM miRNA Isolation kit was usedto enrich the small RNA fraction in these total RNA samples. Librarypreparation using a SOLiD TM Total RNA-Seq kit with modified protocolwas performed on the enriched RNA followed by sequencing onSOLiD TM system. Our results show that small RNA extracted from FFPEsamples was successfully converted to small RNA libraries. Very similarmapping statistics were obtained from matched FFPE and fresh-frozensamples after SOLiD TM sequencing. A good correlation of miRNAexpression pattern was also observed. This suggests that miRNAmolecules are less affected by sample degradation and RNA-proteincrosslink. This study provides a foundation for miRNA expressionanalysis on SOLiD TM system using FFPE samples in cancer and otherdiseases.**169 Structural Insights into theMechanism of microRNA ModulatedViral TranslationS. Patel 2 , E.A. Pham 1 , P. Pang 1 , M.A. Winters 1 ,M. Eckart 2 , J.S. Glenn 1,31Department of Medicine, Stanford UniversitySchool of Medicine, Stanford, CA, United States;2Protein and Nucleic Acid Core Facility, StanfordUniversity School of Medicine, Stanford, CA, UnitedStates; 3 Palo Alto Veterans Administration MedicalCenter, Palo Alto, CA, United StatesMicroRNAs (miRNAs) are small non-coding regulatory RNAs that controla vast array of cellular processes by repressing mRNA translation. LiverexpressedmiR-122 is a miRNA that has been co-opted by hepatitis Cvirus (HCV) to enhance viral translation. Recently, miR-122 antagomirtherapy in non-human primates has been shown to suppress HCVviremia; this proof-of-concept study demonstrates the considerablepotential of this novel antiviral strategy. The mechanism by which miR-122 modulates HCV translation, however, is unclear. To examine thestructural changes that miR-122 exerts on the HCV internal ribosomalentry site (IRES), we developed an advanced Selective 2’-HydroxylAcylation analyzed by Primer Extension (SHAPE) method of analyzingRNA architecture. SHAPE determines RNA secondary structure atsingle-nucleotide resolution, with an accuracy far superior to othermapping methods. Using the above strategy, we show that binding ofmiR-122 to one of its target sites within the 5’ UTR of HCV induces aconformational shift in the HCV IRES at the distant AUG translation startsite. Surprisingly, binding of miR-122 to its second target site in HCVis mediated by a number of non-canonical base-pairings. Mutation ofthe 3’ half of miR-122 (tail) disrupted these non-canonical interactionsand its ability to induce a conformational shift at the AUG start site.We also observed that, in vitro, the first miR-122 target site in HCVis part of a putative triple-strand RNA motif. These results providethe first demonstration that the tail of this liver-encoded miRNA candirectly alter the RNA conformation of the HCV IRES, and therebyprovide new insights into the mechanism by which miR-122 influencesviral translation. We also show that the 5’ UTR of HCV contains a triplexstructure important for viral translation.Poster AbstractsABRF 2011 — Technologies to Enable Personalized Medicine • 79