At a glance • Profiling of microRNAs in 96 well and 384 well plates using just 20ng total RNA or 35 µl serum/plasma to profile hundreds of microRNAs – no pre-amplification necessary • Unmatched sensitivity – reliable quantification of individual microRNAs from as little as 1pg total RNA • Superior specificity – validated LNA™-enhanced primers enable specific quantification of microRNAs that differ by a single nucleotide • Easy to use – protocol completed in 3 hours • Based on SYBR ® Green – enables quality control by melt curve analysis • Step-by-step directed full data analysis solution using Exiqon GexEx A unique system for microRNA profiling The miRCURY LNA™ Universal RT microRNA PCR offers the best available combination of performance and ease-of-use on the microRNA real-time PCR market because it unites two important features (Figure 1 & 2): • Universal RT – One first-strand cDNA synthesis reaction; the RT reaction can be used as template for multiple microRNA real-time PCR assays. • LNA™ PCR amplification – Both PCR amplification primers (forward and reverse) are microRNA-specific and optimized with LNA™. The result is 1) exceptional sensitivity as well as extremely low background enabling accurate quantification of very low microRNA levels and 2) highly specific assays that allow discrimination between closely related microRNA sequences (Figure 3 & Table 1). Product coverage We offer solutions both for microRNA expression profiling and for quantification of individual microRNAs (Overview on next page): • miRNome panels – contain pre-aliquoted PCR primer sets in 384-well PCR plates. Just add cDNA and PCR master mix to the wells and run your real-time PCR profiling. Two panels covering 742 human microRNAs and two panels covering 748 mouse and rat microRNAs are available. Several reference genes and controls are included in all panels. Compatible with most real-time PCR instruments. • Focus panels – 96 well and 384 well plates with microRNA assays selections conveniently optimized for serum/plasma samples and cancer research. Clinically proven platforms. • Pick-&-Mix panels – design your own panels from our wide selection of primer sets and six plate layouts. These panels are ideal for investigating or validating microRNA signatures and subsets on a medium to large number of samples. • Individual assays – validated real-time PCR primer sets for quantification of human, mouse and rat microRNAs and 12 endogenous reference genes are available. Custom assays can be designed for any microRNA using online tool. • Other reagents needed for first-strand cDNA synthesis and real-time PCR: the miRCURY LNA™ Universal RT cDNA synthesis kit and the miRCURY LNA™ SYBR ® Green master mix. All panels are delivered in a ready-to-use format with 10 µL reaction volume per well. Figure 1. Schematic outline of the miRCURY LNA™ Universal RT microRNA PCR System. A polyA tail is added to the mature microRNA template (step 1A). cDNA is synthesized using a PolyT primer with a 3’ degenerate anchor and a 5’ universal tag (step 1B). The cDNA template is then amplified using microRNA-specific and LNA™-enhanced forward and reverse primers (step 2A). SYBR ® Green is used for detection (step 2B). Figure 1 Step 1: First-strand synthesis (RT) Step 2: Real-time PCR amplification Mature microRNA AAAAAAAAAAAAAAAAAAAA A) 3’ degenerate anchor miR-specific forward primer miR-specific reverse primer AAAAAAAAAAAAAAAAAAAA TTTTTTTTTTTTTTT TTTTTTTTTTTTTTT 5’ universal tag B) A) B) miRCURY LNA™ Universal RT microRNA PCR Figure 2 Figure 2 . Overview of the miRCURY LNA™ Universal RT PCR workflow. The PCR primer sets have been designed for optimal performance when used with the miRCURY LNA™ SYBR ® Green master mix. Use of other master mixes might affect the quality of the results. Ready-to-use panels cover miRNome, Focus and Pick-&-Mix panels, but can be replaced by individual PCR primer in this workflow. Universal cDNA synthesis kit 3 hours GenEx software Ready-to-use panel + SYBR ® Green master mix RT PCR Data analysis 4 3 2 1 0 -1 Normal Tumor Tumor stroma Tumor Total • miR-21 • let-7a Relative expression (log2) Functional Analysis Localization Isolation Expression Analysis
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At a glance•ProfilingofmicroRNAsin96welland384wellplatesusingjust20ngtotalRNA
A unique system for microRNA profilingThemiRCURYLNA™UniversalRTmicroRNAPCRoffersthebestavailablecombinationofperformanceandease-of-useonthemicroRNAreal-timePCRmarketbecauseitunitestwoimportantfeatures(Figure1&2):• Universal RT –Onefirst-strandcDNAsynthesisreaction;theRTreactioncan
Figure 4. Expression profiling of 742 microRNAs using 40 ng total RNA from tumor and normal FFPE sections. Real-timePCRwasperformedusingtriplicateRTreactionspersampleonready-to-usemicroRNApanelsIandII(742microRNAsintotal).Datafrom424microRNAswithCpvalues<37isincluded.Normalizedexpressionisshownasfoldchangesintumorcomparedtonormal.Outof424microRNAsexpressed,144were>2-folddown-regulatedinthetumor(bluedots)and26were>2-foldup-regulatedinthetumor(reddots).
Product coverage - overview
Figure 3. LNA™-enhanced primers are more sensitive than DNA primers.LNA™PCRprimersfromthemiRCURYLNA™UniversalRTmicroRNAPCRsystemandcommerciallyavailableDNA-basedprimersetswereusedforreal-timePCRamplificationofserialdilutedcDNA(106-10copies).
Figure 3
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Accurate microRNA expression profiling with minimal sampleThemiRCURYLNA™UniversalRTmicroRNAPCRenablesprofilingofsetsofmicroRNAsin384wellplatesusingonly20ngtotalRNA.ThismakesitpossibletodohighqualitymicroRNAexpressionprofilinginsamplesthatcontainverylittletotalRNA,suchasFFPEsections(Figure4)andserum/plasma.Pre-amplificationofthecDNAisnotrequired.
Fast, easy and reproducibleSavetimeandeffortinthelaboratorywiththe3houreasy-to-followprotocol.ByusingthesameRTreactionastemplateinallsubsequentPCRreactions,theprocedureisgreatlysimplifiedcomparedtosystemsthatrequiremicroRNA-specificfirst-strandsynthesis.Thenumberofpipettingstepsisreducedtoaminimumandtechnicalvariationisminimized.Asaresult,itispossibletoachieveextremelyhighreproducibilityfromday-to-dayandevensite-to-site(Figure8).
Reference and control primer setsSeveralreferenceprimersetsareavailableforreliabledatanormalization.Theseprimersetstargetendogenoussmallnon-codingRNAsthatareconstitutivelyexpressedinawidevarietyoftissues(seewww.exiqon.com/mirna-pcrformoredetails).SomemicroRNAscanalsobeusedasreferencegenesandtheonlyreferencegenesavailableinserum/plasmasamples.Itisimportanttoalwaysconfirmtheconsistentandunvaryingexpressionofageneinalltissuesortreatmentsofanexperimentbeforechoosingitasthereference.
A customized data analysis softwareAspecificallyadaptedversionofthecomprehensiveqPCRanalysissoftware,GenEx,isavailable.Anewuserinterfaceallowsgenerationoftemplatesforusewithdifferentreal-timePCRinstrumentsandrapidimportofdatadirectlyintoGenEx.
Figure 5. Accurate quantitation from 1 pg total RNA starting material. Datafromtheamplificationof7microRNAsinserialdilutionsofhumancolon(C)orheart(H)totalRNAareshown.AllmicroRNAassaysexhibitlinearread-outwithcorrelationcoefficientsR(2)>0,99.
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Figure 6. Detection of differential expression of microRNAs in LCM specimens from tissue FFPE sections. Areasbetween8000and13000µm2ofnormaltissue,tumortissueandtumorstromawereisolatedbylaserdissectionfromFFPEsectionsofhumancolon(A).TotalRNAwasextractedandmicroRNAlevelswerequantifiedusingmiRCURYLNA™UniversalRTPCR(B).Normalizeddataareshownonalog2scaleasrelativeexpressioncomparedtonormaltissue.Hsa-miR-103andhsa-let-7awereusedasreferencegenes.
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Table 1
Table 1. Excellent discrimination between closely related microRNA family members. EachPCRassaywastestedagainstsyntheticRNAfromthe8familymembersofthelet-7family.EachassaywasfoundtobeveryspecificforthemicroRNAforwhichitwasdesigned.
Let-7 family templateshsa-let-7a hsa-let-7b hsa-let-7c hsa-let-7d hsa-let-7e hsa-let-7f hsa-let-7g hsa-let-7i
Contact informationOutside North AmericaPhone:+4545650929Fax:+4545661888exiqon.com/contact
exiqon.com/mirna-pcr
North AmericaPhone:(781)376-4150Fax:(781)376-4152exiqon.com/contact
Figure 8. Excellent site-to-site correlation of Ready-to-use microRNA panels. AliquotsofthesameheartandlivertotalRNAwasexpressionprofiledonmicroRNAReady-to-usePCRpanelsintwodifferentlocations(onseparatedaysbydifferentoperators)usingdifferentRocheLightCycler®480Real-TimePCRSystems.ThecorrelationbetweenaverageCqvalues(triplicateRTreactions)fromallmicroRNAswithsignalsbelow35Cqvalues(totalof297datapoints)isshown.
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Figure 7. Specific and sensitive amplification of microRNA.Amplificationcurves(A)andmeltingcurves(B)fromthehsa-miR-145miRCURYLNA™UniversalRTmicroRNAPCRassayperformedonaserialdilutionofhumancolontotalRNA(100ngto1pg)doneintriplicate.Theamplificationcurvesdemonstratesensitiveandreproducibledetectiondownto1pgoftotalRNAinputandnobackgroundsignal.ThemeltingcurvesshowonlyonemajorTmpeakcorrespondingtoawell-definedmeltingtemperature(Tm)oftheamplicondemonstratingspecificamplificationofhsa-miR-145.
