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Michip) Based on Locked Nucleic Acids (LNA JOBNAME: RNA 12#5 2006 PAGE: 1OUTPUT: Saturday April 115:14:46 2006 cshl/RNA/111792/RNA23324 Downloaded from rnajournal.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press METHOD Asensitive array formicroRNA expression profiling (miChip) based on lockednucleic acids (LNA) MIRCOCASTOLDI, 1,3 SABINE SCHMIDT, 2 VLADIMIR BENES, 2 MIKKEL NOERHOLM, 5 ANDREAS E. KULOZIK,1,4 MATTHIAS W. HENTZE, 3,4 and MARTINA U. MUCKENTHALER1,4 1 Department of Pediatric Oncology,Hematology and Immunology, University of Heidelberg, Germany 2 Genomics Core Facility, 3 Gene Expression Unit, and 4 Molecular Medicine Partnership Unit, EMBL, Heidelberg, Germany 5 Research and Development, Exiqon, Vedbaek, Denmark ABSTRACT MicroRNAs represent aclass of short ( ; 22 nt), noncoding regulatory RNAs involved in development, differentiation, and metabolism. We describe anovel microarray platform for genome-wide profiling of mature miRNAs (miChip) using locked nucleic acid (LNA)-modified capture probes. The biophysical properties of LNA were exploited to design probe sets for uniform, high-affinity hybridizations yielding highly accurate signals able to discriminate between single nucleotide differences and, hence, between closely related miRNA family members. The superior detection sensitivity eliminates the need for RNA size selection and/or amplification. MiChip will greatly simplify miRNA expression profiling of biological and clinical samples. Keywords: microRNAs; microarrays; LNA; RNA translation; mouse INTRODUCTION agivencell or tissue (Doench and Sharp 2004;deMoor et al. 2005). The accurate profiling of miRNA expressionthus MicroRNAs (miRNAs) constitute aclass of recently dis- represents an important tooltoinvestigate physiological covered short regulatory RNAs that control gene expression and pathophysiological states. post-transcriptionallyin, e.g., development, differentiation, Different methodologies havebeenused to profile and metabolism (Krichevsky et al.2003; Abbott et al. 2005; miRNA expression, including Northern blotting with Chenand Lodish 2005; de Moor et al. 2005; Harfe 2005; radiolabeled probes(Sempere et al. 2004; Valoczi et al. Leamanetal. 2005). miRNAsare initially transcribed as 2004), oligonucleotidemacroarrays(Krichevsky et al. 2003; long precursorRNA molecules (pri-miRNAs)and succes- Sioud and Rosok 2004), quantitative PCR-based amplifi- sivelyprocessed by Drosha and Dicer complexes into their cation of precursorormature miRNAs(Schmittgen et al. mature forms of z 22 nt. 2004;Jiang et al. 2005; Shi and Chiang 2005),bead-based Cloning efforts and bioinformatic predictions suggest profiling methods(Barad et al. 2004; Lu et al. 2005), and that miRNAsmay regulate up to 20%–25% of mammalian DNA microarrays spotted onto glass surfaces (Babak et al. genes (Lewis et al. 2005). miRNAscontrol RNAexpression 2004;Liu et al. 2004; Miska et al. 2004; Nelson et al. 2004; at the level of turnover and/or translation via base-pairing, Thomson et al. 2004; Baskerville and Bartel2005). By usually to their 3 9 -untranslated regions (Lee et al. 1993; de contrast to mRNA profiling technologies,miRNA profiling Moor et al. 2005). Boththe qualitative and the quantitative must address the short nature ( z 22 nt) of miRNAs and expressions of miRNAs, therefore, are expected to exert should be able to distinguish between miRNAsthat differ aprofound regulatory influence on the transcriptome of by as little as asingle nucleotide. Microarray-basedmiRNA profiling assays constitute an efficientmethodology to screen in aparallel fashion for the Reprint requeststo: Martina U. Muckenthaler, Department of Pediatric expression of alarge number of miRNAsthrough extensive Oncology, Hematology and Immunology, University of Heidelberg, Im NeuenheimerFeld 156, D-69120 Heidelberg, Germany;e-mail: martina. sample collections. While reliable expression profiles can be [email protected]; or Matthias W. Hentze, Gene Ex- obtained on most of the experimental platforms described pression Unit, EMBL, Meyerhofstrasse 1, D-69117 Heidelberg, Germany, so far, their use can be limited by the large amount of e-mail: [email protected]. Article published online ahead of print. Article and publication date are starting material needed (Miska et al. 2004;Thomson et al. at http://www.rnajournal.org/cgi/doi/10.1261/rna.2332406. 2004), work-intensive miRNA enrichmentprocedures (Miska RNA (2006), 12:913–920. PublishedbyCold Spring Harbor Laboratory Press. Copyright Ó 2006 RNA Society. 913 A23324 Castoldi et al. METHOD RA JOBNAME: RNA 12#5 2006 PAGE: 2OUTPUT: Saturday April 115:14:47 2006 cshl/RNA/111792/RNA23324 Downloaded from rnajournal.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press Castoldi et al. et al. 2004; Baskerville and Bartel 2005), and/or amplifica- probes of standard composition. At amedium hybridization tion and labeling protocols that can introduce quantitative temperature(e.g., 65° C), capture probes with lower T m values bias and, hence, experimental error (Barad et al. 2004; Liu will yield lower signals, while capture probeswith higher T m et al. 2004;Miska et al. 2004; Baskerville and Bartel 2005). values will display impaired nucleotide discrimination and, In addition,discrimination between single nucleotide dif- hence, lowerspecificity. ferences is notalwayspossible(Miskaetal. 2004;Baskerville To overcomethese limitations, we spotted atest set andBartel2005). of capture probes on coated glasssurfaces, which consist We describe anovel microarray platform (miChip) that of either unmodified DNA oligonucleotides or of adefined accuratelyand sensitively monitors the expressionof combination of unmodified and LNA-modified nucleotide miRNAswithout prior need for RNA size fractionation analogs (Tolstrup et al. 2003). The LNA modification raises and/oramplification and that can discriminate among the thermostability of nucleic acid duplex by up to 4 ° Cper closely related miRNA family members. The described tech- nucleotide when the complementary strand is DNA, and up nologyisbased on the use of lockednucleic acid (LNA)- to 8 ° Cwhen the complementary strand is RNA (Nielsen et modified oligonucleotidesthat were previously recognized al. 2003). In afirst approach, we compared unmodified and for their superior performance in Northern blotting and LNA-modified capture probe test sets of identical sequen- miRNA-specific in situ hybridization assays (Valoczi et al. ces (see Supplemental Table 1). The capture probes were 2004;Thomsen et al. 2005;Nelson et al. 2006). LNA is a spotted onto N-hydroxysuccinamide(NHS)-coated synthetic RNA/DNA analog characterized by increased (CodeLink, GE Healthcare) slides,which proved to possess thermostability of nucleic acid duplexes when LNA mono- superior properties regardingthe binding of short (20 mer) mers are introduced into oligonucleotides(Braasch and oligodeoxynucleotides in preliminary experiments (see Corey 2001). Supplemental Fig. 1A,B; Supplemental Table 4). We hypothesized that LNA-modified capture probes (1) Arrays were hybridized to total murine liver RNA may result in amore sensitive detection of miRNAs in directly labeledatthe 3 9 -end by ligation of ashort,Cy-dye comparison to unmodified DNA-based capture probes and modified linker sequence (Fig. 1; see Materialsand Methods). (2) can be designed suchthat an uniformmelting temper- The hybridizationconditions were separately optimized for ature ( T m )can be applied to agenome-wide set of miRNAs the immobilized DNA and LNA-modified capture probes, by adjusting the LNA content and the length of the capture respectively. The miRNA profile obtained fromDNA-based probes (Baskerville and Bartel2005). T m normalization of capture probes is similar to the one generated by capture capture probes permits to establish normalized hybridiza- probes including LNA-modified nucleotides and consistent tion conditions suitable for all miRNAs,which cover a with previously published data (Fig. 1; Babak et al. 2004; range of T m sbetween 45° Cand 74° C. Hybridization con- Liu et al. 2004;Sempere et al. 2004; Thomson et al. 2004; ditionsoptimized forhighstringencybindingnot only Baskerville and Bartel 2005). However, LNA-modified cap- accurately detect theexpression of miRNAs butalsoincrease ture probesyield aseveral-fold-increased hybridizationsig- thehybridization specificityfor relatedmiRNA family mem- nal in comparison to the unmodified DNA capture probes bers that oftendifferbyaslittleasasingle nucleotide when the same amount of input RNA is used and identical (Griffiths-Jones 2004). labelingprotocols are employed. The higher sensitivity of the LNA-modified capture probes is most obvious when lower amounts (2.5–5 m g) of total RNA are used. These RESULTS resultsshow that LNA-modified capture probesare more To establishamicroarray platform that accurately and sen- sensitive than DNA capture probes, offering superior per- sitively measures the expressionlevels of miRNAs without formance for miRNA detection. need for RNAsize fractionation and/or RNAamplification, we optimized RNA preparationprotocols, coating chemis- T -normalized capture probes facilitate tries for glass surfaces, chemical modifications of capture m mismatch discrimination probes, as well as labeling and hybridizationprotocols. To benefit from the biophysical properties offeredbythe LNA-modification, capture probes with auniformly nor- Locked nucleic acid (LNA)-modified DNA malized T of 72° Cwere designed. Probe design included oligonucleotides display superior
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