Simultaneous Detection of Different MicroRNA Types Using ... file2 JournalofNucleicAcids (miRNPs)...

Hindawi Publishing CorporationJournal of Nucleic AcidsVolume 2013 Article ID 496425 13 pageshttpdxdoiorg1011552013496425

Research ArticleSimultaneous Detection of Different MicroRNA TypesUsing the ZIP-Code Array System

Sonja U Weishaupt1 Steffen Rupp2 and Karin Lemuth2

1 Institute of Interfacial Process Engineering and Plasma Technology (IGVP) University of Stuttgart Nobelstraszlige 1270569 Stuttgart Germany

2 Fraunhofer Institute for Interfacial Engineering and Biotechnology (IGB) Nobelstraszlige 12 70569 Stuttgart Germany

Correspondence should be addressed to Karin Lemuth karinlemuthdeboschcom

Received 3 March 2013 Accepted 9 May 2013

Academic Editor Ramon Eritja

Copyright copy 2013 Sonja U Weishaupt et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

MicroRNAs (miRNAs) are important negative regulators of gene expression Their implication in tumorigenesis is based on theirdysregulation in many human cancer diseases Interestingly in tumor cells an altered ratio of precursor and mature miRNA levelshas been described Consequently differences in miRNA type levels have a high potential as biomarkers and comparative high-throughput-based detectionmight permit amore accurate characterization of subtypes especially in the case of very heterogeneoustumor entities Several molecular methods exist for the detection of mature and precursor miRNAs DNA microarrays arepredestinated as a high-throughput method for comprehensive miRNA detection in tumors However the simultaneous array-based detection of both these miRNA types is limited because the mature miRNA sequence is identically present in both formsHere we present a ZIP-code DNA microarray-based system in combination with a novel labeling approach which enablesthe simultaneous detection of precursor and mature miRNAs in one single experiment Using synthetic miRNA templates wedemonstrate the specificity of the method for the different miRNA types as well as the detection range up to four orders ofmagnitude Moreover mature and precursor miRNAs were detected and validated in human tumor cells

1 Introduction

MicroRNAs (miRNAs) are small noncoding RNAs that areknown to have important regulatory functions in geneexpression and influence various biological processesmdashlikecell growth differentiation and apoptosis in eukaryotes(reviewed in [1 2]) Because of their involvement in thesebasic cellular processes miRNAs also play an important rolein tumor development (reviewed in [3ndash5]) Genes encodingmiRNAs are located on the chromosome as independenttranscription units separately from previously annotatedgenes either within introns or even within exons [6] SomemiRNA genes are clustered and transcribed as multicistronicprimary transcripts miRNAs in these transcription units areoften but not always related to each other Further not allmiRNAs of the same cluster are active at the same time [7]

miRNA biogenesis starts from an up to several kilobases long primary miRNA transcript (pri-miRNA) in thenucleus that contains a hairpin structure from which themature miRNA is processed Pri-miRNAs are with a fewexceptions transcribed by RNA polymerase II [8 9] Acomplex including the RNase III endonuclease Drosha andthe double-stranded RNA binding domain protein DGCR8further processes them to short hairpin precursor miRNAs(pre-miRNA) 60ndash110 nucleotides (nt) in length [10 11]Pre-miRNAs are exported by Exportin-5 in a Ran-GTP-dependent mechanism to the cytosol [12] where they arefinally processed by a complex containing Dicer anotherRNase III enzyme and TRBPLoquacious to yield the reg-ulatory active 19ndash24 nt long mature miRNAs in the cytosol[13ndash15] Mature miRNAs are incorporated into RNA-inducedsilencing-complex- (RISC-) like ribonucleoprotein particles

2 Journal of Nucleic Acids

(miRNPs) and target specific mRNAs to trigger mRNAdegradation translational repression or both [6 16 17]

miRNA processing is regulated in development differ-entiation and cancer (reviewed in [18]) Changed levelsof mature miRNAs but unchanged levels of pri- and pre-miRNAs are the hallmark of regulated miRNA processingPosttranscriptional regulation ofmiRNAprocessing has beendescribed on the Drosha and Dicer levels [19ndash21]

Interestingly in tumor cells for example in lung cancer[3 22] or primary effusion lymphoma [23] an altered ratio ofprecursor and mature miRNA levels has been described Theprofiling of different miRNA types in parallel is important fora comprehensive cancer classification [23] especially in thecase of very heterogeneous cancersmdashlike lymphomas [24]Consequently differences in miRNA type levels have a highpotential as biomarkers and their detection might allow amore accurate characterization of different tumor subtypes

Currently precursor andmature miRNAs are detected byseveral molecularmethods (eg northern-blot [25ndash27] qRT-PCR [28ndash30] and DNA microarrays [3 31 32]) Northern-blot analysis allows the simultaneous detection of all miRNAtypes in parallel However the method is laborious andlimited in the number of analytes measured in parallel [33]qRT-PCR offers a higher sensitivity for detection but it alsoperforms on low-throughput level [34]

In contrast DNA microarrays are predestinated as ahigh-throughput method for miRNA detection in tumorsHowever commercially available array systems are not ableto detect the different miRNA types simultaneously with-out costly size exclusion (GeneChip miRNA 30 ArrayAffymetrix Santa Clara USA) or approximate data analysis(GenoExplorer microRNA system GenoSensor Corpora-tion Tempe USA) respectively because these array systemsonly detect the mature miRNA target sequence characteristicfor both miRNA types

Here we show aZIP-codeDNAmicroarray-based systemsimilar to Hauser et al [35] that allows the simultaneousdetection of precursor and mature miRNAs in one singleexperiment The array-platform setup uses unique and dis-tinct ZIP-code probes that show no cross-hybridization toany known organisms Specific primers attached to com-plementary ZIPs (cZIPs) are used for the specific labelingreactionThis allows the simultaneous detection of transcriptlevel variations genotypic differences and DNA-proteininteractions [35] We combined this array platform usingconventional DNA oligomers with a novel labeling approachto simultaneously detect precursor and mature miRNAs on aDNA microarray in one single experiment for a set of ninemiRNAs The approach has the potential for genome-widemiR analysis

2 Material and Methods

21 Cell Lines and Chemicals Human cervix carcinomacell line (HeLa) was obtained from the German Collectionof Microorganisms and Cell Cultures (Braunschweig Ger-many) Keratinocyte cell line (HaCaT) was kindly providedby Professor Dr Fusenig (DKFZ Heidelberg Germany)

All chemicals and reagents were obtained from Carl Roth(Karlsruhe Germany) or as indicated and they were of thehighest available purity

22 Cultivation of Human Cell Lines HeLa and HaCaT celllines were cultivated in 75 cm2 cell culture flasks (GreinerFrickenhausen Germany) and split 1 3 by standardmethodsjust before reaching confluence

All cell culture media were purchased from GIBCO(Life Technologies Darmstadt Germany) HeLa cells weremaintained in RPMI 1640 and HaCaT cells in DMEM (highglucose) each supplemented with 10 fetal calf serum (FCS)1 L-glutamine and 1 Penicillin-Streptomycin

All cells were cultivated under standard conditions at37∘C and 5 CO

2and passaged using Ca2+-Mg2+-free media

for HeLa cells and 10x trypsin-EDTA for HaCaT cells

23 Primers and Oligonucleotides All primers and oligonu-cleotides used in this work were purchased from Metabion(Planegg Germany) qPCR primers and T7 promoter oligo-nucleotides were ordered HPLC purified ZIP-code oligonu-cleotides synthetic oligonucleotides and miRNA specificoligonucleotide-cZIPs were ordered desalted

24 Distinct Labeling of Precursor and Mature miRNA

241 Preparation of Synthetic miRNA Templates Using thefollowing procedure synthetic miRNAs with a 51015840-polyA-tailwere prepared similar to a protocol for linear amplification ofmiRNAs published by Mattie et al [36]

A set of 9 miRNAs known to be involved in tumori-genesis was selected and corresponding mature and stem-loop sequences were extracted from miRBase database V18(httpwwwmirbaseorg correspondingmiRBase accessionnumbers see Table 1) Oligonucleotides reverse complemen-tary (rc) to the corresponding miRNA sequences with amaximum length of 80 nucleotides were designed containinga 14-mer poly d(T) sequence and the rc T7 RNA polymerasepromoter sequence at the 31015840-end (Table 1)

From thesemiRNADNAoligonucleotides T7-promoter-linked DNA oligonucleotides were produced therefore10 120583M of T7 promoter oligonucleotides (51015840-TAATACGAC-TCACTATAGGG-31015840) was mixed with 10 120583M of miRNADNA oligonucleotides and denatured for 3min at 95∘Cand strands were annealed at room temperature over nightThese T7-DNAs were used as an input template for in vitrotranscription (IVT)

Individual IVT reactions were performed for each T7-DNA all containing 5x transcription buffer (FermentasSt Leon-Rot Germany) 2mM NTPs (GE HealthcareMunchen Germany) 50U RNase inhibitor (RNase OutInvitrogen Darmstadt Germany) 30U T7 RNA polymerase(Fermentas St Leon-Rot Germany) and 1 120583g of the individ-ual T7-DNA IVT reactions were incubated for 14 hours at37∘C followed by purification of the amplified miRNA withRNeasy Min Elute Cleanup Kit (Qiagen Hilden Germany)according to the manufacturerrsquos instructions Quantificationof RNA amplificates was performed spectrophotometrically

Journal of Nucleic Acids 3

Table 1 Sequences of mature and precursor synthetic miRNA templates Synthetic miRNA templates composed of reverse complementarymiRNA sequence (bold) 14-mer poly d(T) sequence (standard) and reverse complementary T7 RNA polymerase promoter sequence (italic)Synthetic templates of four precursor synthetic miRNAs were adapted to a maximum length of 80 baseslowast

miRBase ID miRBase accessionnumber Sequence (51015840 to 31015840 orientation)

hsa-miR-9-5p MIMAT0000441 TCA TAC AGC TAG ATA ACC AAA GA TTTTTTTTTTTTTTCCCTATAGTGAGTCGTATTA

hsa-mir-9-3 MI0000468 CA CTC ATA CAG CTA GAT AAC CAA AGA GAG AAA CGG GCCTCCTTTTTTTTTTTTTT CCCTATAGTGAGTCGTATTA

hsa-miR-16-5p MIMAT0000069 CGC CAA TAT TTA CGT GCT GCT A TTTTTTTTTTTTTTCCCTATAGTGAGTCGTATTA

hsa-mir-16-1 MI0000070 TAA CGC CAA TAT TTA CGT GCT GCT AAG GCA CTG CTG ACTTTTTTTTTTTTTT CCCTATAGTGAGTCGTATTA

hsa-miR-92a-3p MIMAT0000092 ACA GGC CGG GAC AAG TGC AAT A TTTTTTTTTTTTTTCCCTATAGTGAGTCGTATTA

hsa-mir-92a-1lowast MI0000093 TCA ACA GGC CGG GAC AAG TGC AAT ACC ATA CAG AAA CATTTTTTTTTTTTTT CCCTATAGTGAGTCGTATTA

hsa-miR-100-5p MIMAT0000098 CAC AAG TTC GGA TCT ACG GGT T TTTTTTTTTTTTTTCCCTATAGTGAGTCGTATTA

hsa-mir-100 MI0000102 TA CCA CAA GTT CGG ATC TAC GGG TTT GTG GCA ACA GGTTTTTTTTTTTTTT CCCTATAGTGAGTCGTATTA

hsa-miR-19b-3p MIMAT0000074 TCA GTT TTG CAT GGA TTT GCA CA TTTTTTTTTTTTTTCCCTATAGTGAGTCGTATTA

hsa-mir-19b-1lowast MI0000074 C AGT CAG TTT TGC ATG GAT TTG CAC AGC AGA ATA TCA CTTTTTTTTTTTTTT CCCTATAGTGAGTCGTATTA

hsa-miR-139-5p MIMAT0000250 CTG GAG ACA CGT GCA CTG TAG A TTTTTTTTTTTTTTCCCTATAGTGAGTCGTATTA

hsa-mir-139 MI0000261 AC ACT GGA GAC ACG TGC ACT GTA GAA TAC ACTTTTTTTTTTTTTT CCCTATAGTGAGTCGTATTA

hsa-miR-29a-3p MIMAT0000086 TAA CCG ATT TCA GAT GGT GCT A TTTTTTTTTTTTTTCCCTATAGTGAGTCGTATTA

hsa-mir-29alowast MI0000087 ATA ACC GAT TTC AGA TGG TGC TAG AAA ATT ATA TTG ACTTTTTTTTTTTTTT CCCTATAGTGAGTCGTATTA

hsa-miR-199b-5p MIMAT0000263 GAA CAG ATA GTC TAA ACA CTG GG TTTTTTTTTTTTTTCCCTATAGTGAGTCGTATTA

hsa-mir-199blowast MI0000282 C CTG AAC AGA TAG TCT AAA CAC TGG GTA GAC GGA GTG GAGTTTTTTTTTTTTTT CCCTATAGTGAGTCGTATTA

hsa-miR-99a-5p MIMAT0000097 CAC AAG ATC GGA TCT ACG GGT T TTTTTTTTTTTTTTCCCTATAGTGAGTCGTATTA

hsa-mir-99a MI0000101 C ACC ACA AGA TCG GAT CTA CGG GTT TAT GCC AAT GGGTTTTTTTTTTTTTT CCCTATAGTGAGTCGTATTA

(V-630 Jasco Gross-Umstadt Germany) 50 ng of eachsynthetic miRNA template was used for cDNA preparationand distinct fluorescent labeling (see Section 244 cDNApreparation and distinct fluorescent labeling)

242 miRNA Isolation and Amplification from Human CellsmiRNA lt200 nt from cell lines was isolated from 107 cellsusing mirPremier microRNA Isolation Kit (Sigma-AldrichTaufkirchen Germany) according to the manufacturerrsquosinstructions for small RNA isolation from mammalian cellcultures Isolated miRNA was analyzed by capillary elec-trophoresis (Agilent Small RNAkit 2100 Bioanalyzer Agilent

Technologies Waldbronn Germany) and RNA concentra-tion and quality were determined photometrically (Nan-oDrop 2000c Peqlab Erlangen Germany or V-630 JascoGross-Umstadt Germany) Only RNA with 260280 nmratios of 18 to 20 was used for reverse transcription andlabeling

Amplification of miRNA was performed using NCodemiRNA Amplification System (Invitrogen Darmstadt Ger-many) according to the manufacturerrsquos protocol startingfrom 30 ng enriched miRNA The in vitro transcriptionreactions were carried out for 16 h followed by purification ofthe amplified 51015840- and 31015840-polyA-tailedmiRNAmolecules usingRNeasy MinElute Cleanup Kit (Qiagen Hilden Germany)


Table 2 Sequences of ZIP oligonucleotides and miRNA-specific oligonucleotides-cZIP Sequences of ZIP oligonucleotides (immobilized onDNA microarray) and miRNA-specific oligonucleotides with cZIP (miRNA-specific sequences are illustrated in bold)

Internal ZIP ID MSO-cZIPset

Sequence of ZIP oligonucleotideon DNA microarray (51015840 to 31015840orientation)

Sequence of miRNA-specific oligonucleotide-cZIP (MSO-cZIP) (51015840 to 31015840orientation)

mi-9 ZIP A A GCAGTGCTCACCGTCCGCGA TCGCGGACGGTGAGCACTGC TCATACAGCTAGATAACCAAAGAmi-9 ZIP B B ACGCGACGCACCTGCTCCAA TTGGAGCAGGTGCGTCGCGT TCATACAGCTAGATAACCAAAGAmi-16 ZIP C A CGGAGTGGCACCAGCGGGAA TTCCCGCTGGTGCCACTCCG CGCCAATATTTACGTGCTGCTAmi-16 ZIP D B GTGGGAGCGACGCAGGGCAG CTGCCCTGCGTCGCTCCCAC CGCCAATATTTACGTGCTGCTAmi-92 ZIP E A TGGCGGTCTGCTGAGCGGTC GACCGCTCAGCAGACCGCCA ACAGGCCGGGACAAGTGCAATAmi-92 ZIP F B GCCTCGAGCCAACACCGCCT AGGCGGTGTTGGCTCGAGGC ACAGGCCGGGACAAGTGCAATAmi-100 ZIP G A TGGCCGGACAGGAGACACGC GCGTGTCTCCTGTCCGGCCA CACAAGTTCGGATCTACGGGTTmi-100 ZIP H B GCCTGCCTTCACGAGCCCAA TTGGGCTCGTGAAGGCAGGC CACAAGTTCGGATCTACGGGTTmi-19b ZIP I A TGGCCGAGACTGCAGGAGCG CGCTCCTGCAGTCTCGGCCA TCAGTTTTGCATGGATTTGCACAmi-19b ZIP K B ACGCCCTCCCAACCTCACGC GCGTGAGGTTGGGAGGGCGT TCAGTTTTGCATGGATTTGCACAmi-139 ZIP L A AGCGGACGACTGCGGACGAG CTCGTCCGCAGTCGTCCGCT CTGGAGACACGTGCACTGTAGAmi-139 ZIP M B TGGCGAGCGCAGTGGCAGAC GTCTGCCACTGCGCTCGCCA CTGGAGACACGTGCACTGTAGAmi-29a ZIP N A AGCGTGCTGGTCGTGGGCCT AGGCCCACGACCAGCACGCT TAACCGATTTCAGATGGTGCTAmi-29a ZIP O B AGCGCCAAGGGTCCTCGGGT ACCCGAGGACCCTTGGCGCT TAACCGATTTCAGATGGTGCTAmi-199b ZIP P A GGGTGGTCCGGAGCGAGCAG CTGCTCGCTCCGGACCACCC GAACAGATAGTCTAAACACTGGGmi-199b ZIP Q B CCTCAGACGGGTAGCGGCGA TCGCCGCTACCCGTCTGAGG GAACAGATAGTCTAAACACTGGGmi-99a ZIP R A CCTCGTCCGTCCGGTCACCA TGGTGACCGGACGGACGAGG CACAAGATCGGATCTACGGGTTmi-99a ZIP S B GGGTGTGGACGCGGAAGCAG CTGCTTCCGCGTCCACACCC CACAAGATCGGATCTACGGGTT

according to themanufacturerrsquos protocol 15 120583g of this ampli-fied totalmiRNAwas used for cDNApreparation and distinctfluorescent labeling (see Section 244 cDNA preparation anddistinct fluorescent labeling)

RNA concentration and quality were determined photo-metrically (NanoDrop 2000c Peqlab Erlangen Germany orV-630 Jasco Gross-Umstadt Germany)

243 miRNA-Specific cZIP-Code Design Oligonucleotidesreverse complementary (rc) to the corresponding maturemiRNA sequences were extended 51015840 with D-DNA cZIPsequences [35] (Table 2)

Each mature miRNA specific oligonucleotide (MSO) waslinked to two different cZIP-code sequences that is twocombinations of mature miRNA specific oligonucleotide-cZIPs (MSO-cZIP) for every miRNA were available

244 cDNA Preparation and Distinct Fluorescent LabelingcDNA synthesis was performed via reverse transcriptionby incorporation of different fluorescence labeled deoxynu-cleotides (Cy3-ATP or Cy3-dUTP)

As starting material for cDNA synthesis and distinctfluorescent labeling of synthetic miRNA 50 ng synthetictemplates of each miRNA were pooled separately for matureand precursor miRNAs

As starting material for cDNA synthesis and distinctfluorescent labeling of mature or precursor miRNA fromcells 15120583g of amplified total-miRNA was used respectively

Labeling with Cy3-dUTP and Cy3-dATP was per-formed individually in two distinct reaction cups as follows025 pmol of each MSO-cZIP (either A or B see also Table 2)was added to a volume of 10 120583L followed by RNA denatu-ration for 5min at 70∘C and annealing of the MSO-cZIPs totheir corresponding miRNAs for 30min at 50∘C

For labeling with Cy3-dUTP the annealed miRNA tem-plate was combined in a final 20120583L reaction mix containing5x reaction buffer (Fermentas St Leon-Rot Germany)05mM dTTP (Qiagen Hilden Germany) 20U RNase Out(Invitrogen Darmstadt Germany) 1875mM MgCl

2(Invit-

rogen Darmstadt Germany) 40U M-MuLV Reverse Tran-scriptase (Fermentas St Leon-Rot Germany) and 005mMCy3-dUTP (GE Healthcare Munchen Germany)

For labelingwithCy3-dATP 05mMof each dCTP dGTPand dTTP and 008mM dATP (Qiagen Hilden Germany)were used instead of 05mM dTTP Further 005mM Cy3-dATP (Perkin Elmer Waltham Massachusetts USA) wasused instead of 005mM Cy3-dUTP

The reverse transcription reaction was performed for 15hours at 37∘C and subsequently heat inactivated at 70∘Cfor 10min After adding 2U Ribonuclease H (InvitrogenDarmstadt Germany) to each sample to degrade RNA andincubation for 20min at 37∘C samples were unified followedby column purification of cDNA by QIAquick NucleotideRemoval Kit (Qiagen Hilden Germany) cDNA was elutedwith 315120583L elution buffer

25 Preparation of Genomic DNA Genomic DNA was ex-tracted from HaCaT cell pellets containing 107 cells using


AllPrep DNARNA Mini Kit (Qiagen Hilden Germany)according to the protocol ldquoGenomic DNA Purification Proto-col from Animal Cellsrdquo DNA concentration and quality weredetermined photometrically

26 ZIP-Code DNAMicroarrays

261 Spotting D-DNA ZIP-code oligonucleotides (Table 2)[35] with 31015840 Amino-C7 linker (50120583M in spotting buffer 13(vv) Nexterion Spot ISpot III (Schott Mainz Germany))were spotted in 16 replicates onto epoxy-coated glass slides(Nexterion Slide E Schott Mainz Germany) using a Micro-Grid II spotter (Digilab Holliston USA)

Slideswere postprocessed andprehybridized according tothe manufacturerrsquos instructions

262 Hybridization Scanning and Data Analysis 135 120583Lhybridization buffer (4x SSC and 01 SDS) was added to315 120583L cDNA solution Hybridization was performed usingLifterSlips (Erie Scientific Anaheim USA) for 16 h at 65∘Cin a hybridization chamber Slides were washed according tothe manufacturerrsquos instructions and scanned with an AxonGenePix 4300A scanner (Molecular Devices SunnyvaleUSA) with a resolution of 10 120583m laser power of 100 andPMT values ranging from 500 to 800 Spot quantification wasperformed usingGenePix Pro 7 software (Molecular DevicesSunnyvale USA) DNA microarray data analysis was basedon averaged background-corrected median values obtainedfrom GenePix Pro 7 software

263 Quantitative Real-Time PCR To validate ZIP-codearray results quantitative real-time PCR (qRT-PCR) ofmature and precursor miRNAs of HeLa cells was performed

Isolated enriched miRNA was treated with AmbionTURBO DNase (Life Technologies Darmstadt Germany) toremove genomic DNA Therefore up to 500 ng small RNAwas mixed in a final volume of 50120583L with 10x Turbo Bufferand 06UTurboDNase I After incubation at 37∘C for 30minmiRNA was purified using RNA Clean amp Concentrator-5 (Zymo Research Irvine USA) according to the manu-facturerrsquos instructions This kit allows the purification andconcentration of RNA gt17 nt

100ndash500 ng ofmiRNAwas reverse transcribed into cDNAvia miScript II RT Kit (Qiagen Hilden Germany) accordingto the manufacturerrsquos instructions using 5x miScript HiFlexBuffer which enables conversion of mature as well as precur-sor miRNA

For qPCR specific primers for individual miRNAs wereused either from commercial miScript Assays (QiagenHilden Germany) [37] or self-designed primers in a finalconcentration of 01120583M (see Table 3) Self-designed primerswere adjusted to a length of 20ndash25 nt and a melting tem-perature of 60∘C plusmn 1∘C using ApE software v117 Primerswere designed and inspected with OligoAnalyzer 31 (IDT) toexclude self-dimers or oligo secondary structures The speci-ficity of primer pairs was checked using Basic Local Align-ment Search Tool (BLAST httpblastncbinlmnihgov)Specificity of qPCRassayswas additionally validated using gel

1 2 3 4 5 6 7 8 9 10 11 12 13

(bp)

500400300

200

100

50

Figure 1 Agarose gel electrophoresis of qRT-PCR amplicons qRT-PCR products were separated in a 3 agarose1x TAE gel at 120Vfor 1 h and stained with ethidium bromide Lane 1 100 bp marker(Fermentas St Leon-Roth Germany) lane 2 50 bp marker (NEBIpswich USA) lane 3 miR-99a lane 4 pre-miR-199b lane 5 pre-miR-9 lane 6 100 bpmarker lane 7 pre-miR-16 lane 8miR-16 lane9 100 bp marker lane 10 miR-9 lane 11 pre-miR-99a lane 12 pre-miR-199b and lane 13 100 bp marker

electrophoresis (3 agarose (Carl Roth Karlsruhe Germany)and 1x TAE buffer (Tris acetate 40mMEDTA 1mM pH 80each Carl Roth Karlsruhe Germany) 120V 1 h) Only theexpected amplicon length (either mature or precursor) foreach primer pair could be detected (see Figure 1) Furtherthe specificity of commercial assays has been confirmed byQiagen

The QuantiTect SYBR Green PCR Kit (Qiagen HildenGermany) was used for qPCR following protocols Real-Time PCR for Detection of Mature miRNA or NoncodingRNA and Real-Time PCR for Detection of Precursor miRNArespectively

qRT-PCR reactions were performed in Light Cycler 480multiwell plates 96 in a volume of 25 120583L with an inputamount of 3 ng cDNA from reverse transcription reaction fordetection of mature miRNA and 10 ng cDNA for precursorsrespectively All assays were performed at least in duplicatesand no template-controls (NTC) were included using waterinstead of cDNA as negative control qPCRwas performed ona LightCycler 480 Instrument (Roche Mannheim Germany)according to the following PCR protocol 95∘C for 15min45 cycles of 94∘C for 15 s 55∘C for 30 s and 70∘C for30 s followed by a melting curve analysis (65∘C to 97∘C)for determination of specificity of amplification Furtherspecificity of qPCR products was validated using agarose gelelectrophoresis (see Figure 1)

Standard curves for determination of the amplificationefficiency of each assay were generated using a dilution seriesof cDNA fromHeLa (in the range of 100 and 10minus4) or genomicDNA from HaCaT cells (24 times 105ndash24 times 101 copies120583L)respectively For PCR efficiency of individual primer pairsplease see Table 3

3 Results and Discussion

We established a method for the simultaneous detectionand distinction of mature and precursor miRNAs in one


Table 3 miRNA-specific primers and assays used in quantitative real-time PCR miRNA-specific commercial assays (Qiagen) and sequencesof self-made miRNA-specific primers used in qRT-PCR (Tm = melting temperature)

miRBase ID MiScript Assay (Qiagen) Forward primerreverse primer (51015840 to 31015840orientation)

Length(nt)

Tm(∘C)

Ampliconlength (bp)

PCRefficiency

Errorrate ()

hsa-miR-9-5p Hs miR-9 1 miScriptPrimer Assay 85ndash87 23 490

hsa-mir-9-3 GAGGCCCGTTTCTCTCTTTGG 21 59 63 16 391CTAGCTTTATGACGGCTCTGTGG 23 59


hsa-mir-16-1 GCAGCACGTAAATATTGGCGTTAAG 25 59 74 18 061GTCAACCTTACTTCAGCAGCACAG 24 60

hsa-miR-199b-5p Hs miR-199b 1 miScriptPrimer Assay 85ndash87 25 347

hsa-mir-199bHs mir-199b 1 PRmiScript PrecursorAssay

89 19 045

hsa-miR-99a-5p Hs miR-99a 2 miScriptPrimer Assay 85ndash87 19 134

hsa-mir-99aHs mir-99a 1 PRmiScript PrecursorAssay

72 19 415

single experiment based on an established ZIP-code DNAmicroarray system with universal probes [35] and a novellabeling procedure For this purpose the miRNAs had to beprepared as follows

Starting with an initial linear amplification of iso-lated miRNA [36] amplified miRNA (amiR) moleculeswith 51015840- and 31015840-polyA-tails were generated On average442 ng plusmn 150 ng amiR could be generated from 30 ng of iso-lated miRNA

The amiR 51015840-polyA-tail of mature amiR was labeled byincorporation of Cy3-dUTP in an enzymatic reaction con-taining only Cy3-dUTP and unlabeled dTTP (Figure 2(a))In this case precursor amiR will not be labeled because of thesubsequent bases following the mature sequence Howeverone can get a false-positive signal if the first subsequent baseis an A In this case the MSO should be extended by a 31015840-T toavoid this

In the case of precursor amiR the precursor sequencefollowing the MSO sequence was labeled using Cy3-dATPand all four unlabeled deoxynucleotides (Figure 2(b)) in anindividual reaction Accordingly mature amiR cannot belabeled on its polyA-tail in this reaction

In the specific amiR labeling reactions MSOs foreach miRNA linked to two different cZIP-code sequences(MSO-cZIPs) were used The ZIP-code part of the MSO-cZIPs allowed their distinction by the subsequent address-ing to individual positions on the ZIP-code microarray(Figure 2(c))

To ensure the comparability of the two distinct labelingapproaches we looked for differences caused by the ZIP-code pairs and differences caused by the different fluo-rescently labeled deoxynucleotides (Cy3-dATP Cy3-dUTP)

We tested ZIP-code pair-miRNA combinations for specificityas described in the next sections

Synthetic templates were used in combination with twodifferent MSO-cZIP sets A and B respectively (Table 2) Allarray experiments were performed in triplicates ZIP-codepair-miRNA combinations were regarded as accurate whenno significant differences within the standard deviations ofthe fluorescent signals could be detected Additionally nounspecific signals should occur on theDNAmicroarray OnlyZIP-code pair-miRNA combinations that pass these criteriaare useful for distinction of mature and precursor miRNA Inthat case detectable differences on the DNA microarray aredue to differences in miRNA type levels only

The specificity of the ZIP-code array for a clear distinctionof different miRNA types was examined by exclusive labelingof mature but not precursor synthetic miRNA and of pre-cursor but not mature synthetic miRNA respectively and bycompetitive hybridization thereafter

To test for cross-hybridization in a first approach labelingof mature synthetic miRNA was performed with Cy3-dUTPand unlabeled dTTP usingMSO-cZIP set A whereas labelingof precursor synthetic miRNA was performed exclusivelywith unlabeled deoxynucleotides and MSO-cZIP set B Thehybridization resulted in a specific detection of maturemiRNA in 100 of all cases as shown in Figure 3(a) No cross-hybridization could be detected

In a second approach labeling of precursor syntheticmiRNA was performed with Cy3-dATP and unlabeled deox-ynucleotides using MSO-cZIP set B and labeling of maturesynthetic templateswas performed exclusivelywith unlabeleddeoxynucleotides and MSO-cZIP set A This hybridizationexhibited no significant signals for mature miRNA and


miRNA specific oligonucleotide A

Mature miRNA

cZIP X

Cy3-dUTP +

dTTP

3998400

5998400

5998400

TTUTTTUTTTUTT

AAAAAAAAAAAAAA

(a)

Precursor miRNA

cZIP Y

Cy3-dATP+

dATPdCTPdGTPdTTP

Precursor sequence Precursor sequence Mature sequence


3998400

5998400

5998400

GATGATTCCGA

AAAAAAAACUGGCUACUAAGGCU

(b)

Precursor miRNA(cZIP Y) Mature

miRNA(cZIP X)

ZIP Y

ZIP X

(c)

Figure 2 Labeling method for mature (a) and precursor (b) miRNA and their specific detection on ZIP-code array (c) Mature miRNAis labeled at the polyA-tail with Cy3-dUTP and unlabeled dTTP using miRNA-specific cZIP-Primer X precursor miRNA is labeled at theconsecutive precursor sequence with Cy3-dATP and unlabeled deoxynucleotides using miRNA-specific cZIP-Primer Y The detection anddifferentiation of mature and precursor miRNA are based on the hybridization of the corresponding ZIP-code parts of the miRNA-specificcZIP-Primers to their complementary ZIP-code sequences immobilized at defined positions on the ZIP-code array

confirms the specificity of the method for exclusive detectionof precursor miRNA (see Figure 3(b))

The threshold for significant signals was set to 190 aubased on unspecific background signal intensity (averagedacross three experiments) (see Figure 3(a))

Additionally specificity studies were performed usingvarying combinations of different synthetic mature andprecursor miRNA templates Labeling of mature miRNAwith Cy3-dUTP and unlabeled dTTP using MSO-cZIP setA and precursor miRNA with Cy3-dATP and unlabeleddeoxynucleotides using MSO-cZIP set B led to the specificpattern expected (data not shown) These results confirmedthe specificity of the system for distinct detection of matureand precursor miRNAs

The distinct detection of precursor and mature miRNAsusing the ZIP-code array is based on the same miRNAsequence coupled to two different ZIP-code oligos anddistinct labeling To ensure that the different ZIP-codesequences of the appropriate ZIP-code pairs do not leadto significant signal differences identical miRNA synthetictemplates were labeled using corresponding miRNA-specificoligonucleotides in two different cZIP combinations

Mature miRNA synthetic templates were reverse tran-scribed with Cy3-dUTP and unlabeled dTTP using MSO-cZIP set A and MSO-cZIP set B respectively The exper-iments were performed in separate Eppendorf cups Afterthe simultaneous hybridization of both solutions to the ZIP-code array signal intensities of corresponding ZIPs for eachmiRNA were compared (see also Figure 4(a)) Comparable

signal intensities could be achieved for seven of the nine ZIP-code pairs Only for ZIP-code pairs ZIP AB and ZIP IKdifferences in signal intensities beyond the standard deviationcould be detected However the absolute signal intensitieswere in a comparable range and therefore these ZIP-codepairs were also included in the following experiments

The same experimental design was set up with precursorsynthetic miRNA templates using Cy3-dATP and unlabeleddeoxynucleotides for labeling (see also Figure 4(b)) In thisexperiment except for ZIP pair IK comparable signalintensities could be detected in all cases

The concordant results indicate that in the case of ZIP-code pair IK variances might be based on variable ZIPbinding efficiencies of the in silico designed D-DNA cZIPsIn these cases respective ZIPs must be excluded from themicroarray setup

To ensure the comparability of the two fluorescentdeoxynucleotides for distinct labeling miRNA synthetictemplates were labeled using corresponding miRNA-specificoligonucleotides in two different cZIP combinations Signalintensities are regarded as comparable if signals can bedetected for both labeling variants (signal to noise ratio gt 3)Mature synthetic miRNAs were labeled with Cy3-dUTP anddTTP using MSO-cZIP set A precursor synthetic miRNAswere reverse transcribed with Cy3-dATP and unlabeleddeoxynucleotides using MSO-cZIP set B

Further synthetic miRNA templates were labeled theother way round resulting in mature synthetic miRNAlabeled with MSO-cZIP set B and precursor synthetic


100

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Gm

i-19b

ZIP

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Lm

i-29a

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Dm

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i-92

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Em

i-100

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Gm

i-19b

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i-139

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Lm

i-29a

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i-199

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Bm

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Dm

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Fm

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i-19b

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Km

i-139

ZIP

Mm

i-29a

ZIP

Om

i-199

bZI

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mi-9

9aZI

PS

(b)

Figure 3 Exclusive detection of mature (a) and precursor (b)miRNA Log signal intensities from three hybridizations of fluo-rescently labeled MSO-cZIPs with the ZIP-code array after specificlabeling of synthetic miRNA templates are presented (a) Specificlabeling of mature synthetic miRNA templates with Cy3-dUTPand unlabeled dTTP using MSO-cZIP set A and precursor syn-thetic miRNA templates were reverse transcribed with unlabeleddeoxynucleotides and MSO-cZIP set B (b) Specific labeling ofprecursor synthetic miRNA templates with Cy3-dATP and unla-beled deoxynucleotides usingMSO-cZIP set B andmature synthetictemplates were reverse transcribedwith unlabeled deoxynucleotidesandMSO-cZIP set A ZIPs for mature miRNA detection A C E GI L N P and R ZIPs for precursor miRNA detection B D F H KM O Q and S

miRNAs with MSO-cZIP set A respectively Comparablesignal intensities for corresponding mature and precursormiRNA were detected for all sets (see also Figures 5(a) and5(b) resp) This allows the combination of both labelleddeoxynucleotides for the simultaneous detection of matureand precursor miRNAs in one DNA microarray experiment

The detection range of the system was estimated usingmiRNA synthetic templates in a range of four orders ofmagnitude as input for different labeling reactions

Mature miRNA synthetic templates were labeled withCy3-dUTP and unlabeled dTTP using MSO-cZIP set Awhereas precursor miRNA synthetic templates were labeled

1

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00ZI

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00ZI

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9bZI

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9bZI

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39ZI

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39ZI

PM

mi-2

9aZI

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mi-2

9aZI

PO

mi-1

99b

ZIP

Pm

i-199

bZI

PQ

mi-9

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PS

(a)

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signa

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6ZI

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PE

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2ZI

PF

mi-1

00ZI

PG

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00ZI

PH

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9bZI

PI

mi-1

9bZI

PK

mi-1

39ZI

PL

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39ZI

PM

mi-2

9aZI

PN

mi-2

9aZI

PO

mi-1

99b

ZIP

Pm

i-199

bZI

PQ

mi-9

9aZI

PR

mi-9

9aZI

PS

(b)

Figure 4 Comparison of different cZIPs linked to identical miRNAoligonucleotides based on synthetic mature (a) and precursor (b)miRNA template labeling Using identical miRNA-specific oligonu-cleotides with different cZIPs in separate Eppendorf cups maturesynthetic miRNA templates were labeled with Cy3-dUTP andunlabeled dTTP (a) or precursor synthetic miRNA templates werelabeled with Cy3-dATP and unlabeled deoxynucleotides (b) Logsignal intensities from subsequent hybridization to the ZIP-codearray in triplicates are shown Different cZIPs for identical miRNAare illustrated contiguous in identical colors

with Cy3-dATP and unlabeled deoxynucleotides usingMSO-cZIP set B

At first mature miRNA synthetic templates were varied(10-fold dilution series with 03ndash0003 pmoles) while the pre-cursor miRNA synthetic template amount was kept constantat 3 pmoles Experiments were performed in triplicates

For all ZIP-code miRNA combinations a minimumdetection range over 2-3 orders of magnitude could beshown (see Figure 6(a)) Five ZIP-codemiRNAcombinations(mi-16 mi-100 mi-139 mi-199b mi-99a) exhibited even adetection range over three orders of magnitude from 003 upto 3 pmoles


1

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ZIP

Am

i-9ZI

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6ZI

PC

mi-1

6ZI

PD

mi-9

2ZI

PE

mi-9

2ZI

PF

mi-1

00ZI

PG

mi-1

00ZI

PH

mi-1

9bZI

PI

mi-1

9bZI

PK

mi-1

39ZI

PL

mi-1

39ZI

PM

mi-2

9aZI

PN

mi-2

9aZI

PO

mi-1

99b

ZIP

Pm

i-199

bZI

PQ

mi-9

9aZI

PR

mi-9

9aZI

PS

(a)

1

10

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1000

10000

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ZIP

Am

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6ZI

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6ZI

PD

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2ZI

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2ZI

PF

mi-1

00ZI

PG

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00ZI

PH

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9bZI

PI

mi-1

9bZI

PK

mi-1

39ZI

PL

mi-1

39ZI

PM

mi-2

9aZI

PN

mi-2

9aZI

PO

mi-1

99b

ZIP

Pm

i-199

bZI

PQ

mi-9

9aZI

PR

mi-9

9aZI

PS

(b)

Figure 5 Comparison of mature and precursor miRNA labelingwith different fluorescent deoxynucleotides Log signal intensitiesfrom three hybridizations after labeling of mature synthetic miRNAwith Cy3-dUTP and unlabeled dTTP in combination with MSO-cZIP set A (a) or MSO-cZIP set B (b) and precursor syntheticmiRNA with Cy3-dATP and unlabeled deoxynucleotides in combi-nation with MSO-cZIP set B (a) or MSO-cZIP set A (b) are shownComparative cZIPs for each miRNA are illustrated contiguous inidentical colors

In addition mature miRNA synthetic template amountwas kept constant (3 pmoles) in all three hybridizationswhereas precursor miRNA synthetic templates varied in10-fold dilution steps from 03 down to 0003 pmoles Adetection range of the system over three orders of magnitudewas confirmed for 7 out of 9 tested ZIP-code pair-miRNAcombinations (for mi-16 mi-100 mi-19b mi-139 mi-29a mi-199b mi-99a) as shown in Figure 6(b) For mi-16 andmi-29aa detection range over four orders of magnitude (from 0003up to 3 pmoles) was observed

Altogether a detection range at least over three ordersof magnitude was achieved using probe sets mi-16 mi-100mi-139 mi-29a mi-199b and mi-99a so these probe setswere proven to be qualified for quantification Regarding theprobe sets for mi-9 mi-92 and mi-19b a qualitative miRNAdetection based on on-off signals is possible

The experimental setup is based on the direct comparisonof mature and precursor miRNA templates and might there-fore possibly be influenced by template quantification biasesNevertheless wewere able to showan average detection rangeof our system over three orders of magnitude for the majorityof cases for twomiRNAs evenup to four orders ofmagnitude

After establishment of the method the assay was appliedto analyze human tumor cells Both mature and precursormiRNAs amplified from HeLa cells were specifically labeledusing the described method Individual signals for matureand precursor amiRs were detected with the ZIP-code array(Table 4 Figure 7)

To confirm the findings from array-based mature andprecursor amiR detection in HeLa cells we used qRT-PCR asan independent method for validation A set of four miRNAswas chosen which passed our expected criteria from theestablishment with synthetic miRNA templates above qRT-PCRwas used to check for on-off signals that is the presenceor absence of corresponding mature and precursor miRNAforms

qRT-PCR amplification curves were generated for themiRNA pairs miR-16 and pre-miR-16 miR-9 and pre-miR-9 miR-99a and pre-miR-99a and miR-199b and pre-miR-199b (for PCR efficiencies please see Table 3) As expectedin melting curve analyses and 3 agarose gel electrophoresisonly the expected amplicon (either mature or precursor) foreach primer pair could be detected (see Figure 1) Ct valuesfrom qRT-PCR are listed in Table 4

In order to determine absence or presence of a particularmiRNA in qRT-PCR assays a threshold Ct value was definedbased on quantification of genomic DNA from HaCaTcell line for pre-miR-16 pre-miR-99a and pre-miR-199b indilution steps from 24 times 105ndash24 times 101 copieswell Copynumbers were calculated according to Lemuth et al [41]A copy number of 24 times 105 24 times 104 24 times 103 and24 times 10

2 corresponded to average Ct values of 217 plusmn 17252 plusmn 22 290 plusmn 20 and 3465 respectively According tothis the sensitivity of the assays was set to 24 times 102 copiescorresponding to a Ct value of 3465 Ct values equal orbelow this threshold indicate presence of pre-miR and miR(Table 4)

In accordance with DNA microarray signal intensitiesthe presence of miR-16 pre-miR-16 miR-99a pre-miR-99amiR-199b and pre-miR-199b in HeLa cells was confirmed byqRT-PCR

Inconsistencies between our array and qPCR results wereonly detected for pre-miR-9 and miR-9 The array dataindicate no presence of mature miR-9 whereas the qPCRindicates limited amounts of mature miR-9 although atlevels close to the threshold Comparing these data to datafrom the literature two independent investigations usingdifferent methods show that mature miR is not expressedin HeLa cells [38 39] This indicates that the qRT-PCR datain this case might give a low but reproducible false-positivesignal For the precursor miR-9 no signal was detectedin the qPCR whereas the array data showed a low butreproducibly detectable signal for pre-miR-9 In this case thePCR efficiencywas the least efficient for allmiR tested Taking


Log

signa

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ensit

y (a

u)

1

10

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1000

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ZIP

A

mi-9

ZIP

B

mi-1

6ZI

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mi-1

6ZI

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2ZI

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2ZI

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mi-1

00ZI

PG

mi-1

00ZI

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mi-1

9bZI

PI

mi-1

9bZI

PK

mi-1

39ZI

PL

mi-1

39ZI

PM

mi-2

9aZI

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mi-2

9aZI

PO

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99b

ZIP

P

mi-1

99b

ZIP

Q

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9aZI

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9aZI

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03 pmoles003 pmoles

0003 pmoles3 pmoles

(a)

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signa

l int

ensit

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u)

1

10

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1000

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03 pmoles003 pmoles

0003 pmoles3 pmoles

mi-9

ZIP

A

mi-9

ZIP

B

mi-1

6ZI

PC

mi-1

6ZI

PD

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2ZI

PE

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2ZI

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00ZI

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00ZI

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9bZI

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mi-1

9bZI

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mi-1

39ZI

PL

mi-1

39ZI

PM

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9aZI

PN

mi-2

9aZI

PO

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99b

ZIP

P

mi-1

99b

ZIP

Q

mi-9

9aZI

PR

mi-9

9aZI

PS

(b)

Figure 6 Simultaneous detection of mature and precursor miRNAs over four orders of magnitude Log signal intensities from threehybridizations of precursor and mature miRNAs in different amounts are shown (a) Mature miRNA synthetic templates variable (03 0030003 pmoles) precursor miRNA synthetic templates constant (3 pmoles each) (b) mature miRNA synthetic templates constant (3 pmoleseach) precursor miRNA synthetic templates variable (03 003 0003 pmoles) ZIPs for mature miRNA detection A C E G I L N P andR ZIPs for precursor miRNA detection B D F H K M O Q and S

these results together into account especially with regard tothe literature data indicates that most likely the qPCR formiR-9 and pre-miR-9 renders the critical results Also the lowsignal intensities might further facilitate false-positive andfalse-negative results qRT-PCR setup of miRNAs are verylimited in placement of primersThis limitation often rendersthe design of accurate qPCR tools a challenging task

The array results for all other testedmaturemiRNAs are incomplete accordance with both our qPCR and the literaturedata (see Table 4)

In summary we have established a method for thesimultaneous detection and differentiation of mature andprecursor miRNA molecules based on a ZIP-code arraysystem

Using synthetic miRNA templates we clearly demon-strated the specificity of the method for the different miRNAtypes as well as a detection range up to four orders ofmagnitude Moreover we applied our method to amiR fromthe cellsThe detection of mature and precursor miRNAs wasvalidated in a human tumor cell line model

Themethod presented has the ability to detect any knownmiRNA sequence This is important as mature miRNA wasfound to be present in different length variants in cells[42ndash44] These miRNA isoforms can alter miRNA targetregulation [43] changing their role in tumorigenesis Ourmethod offers the possibility for the comprehensive detectionof known miRNA variants depending on the availablenumber of ZIPs on the DNA microarray


Table 4 Validation of DNA microarray-based miRNA detection in human cells Mature and precursor miRNA detection in HeLa cells byDNA microarray validation of array signals by comparison to qRT-PCR data andor comparison to literature data for mature miRNAs Thepresence of individual miRNAs (Figure 7) was determined based on array signal intensity (threshold 190 au according to Figure 3(a)) orqRT-PCR Ct values equal or below a threshold Ct value of 3465 (corresponding to 24 times 102 copies)

miRBase ID Internal ZIP ID MatureprecursormiRNA

Presence of miRNAaccording to DNAmicroarray results

Presence of miRNA accordingto qRT-PCR (average Ct value

from triplicates)

Presence of miRNAaccording to Reference

hsa-miR-9-5p mi-9 ZIP A Mature No Yes (331) No [38 39]hsa-mir-9-3 mi-9 ZIP B Precursor Yes No (gt400)hsa-miR-16-5p mi-16 ZIP C Mature Yes Yes (181)hsa-mir-16-1 mi-16 ZIP D Precursor Yes Yes (346)hsa-miR-92a-3p mi-92 ZIP E Mature No nd No [39]hsa-mir-92a-1 mi-92 ZIP F Precursor Yes nd nahsa-miR-100-5p mi-100 ZIP G Mature Yes nd Yes [40]hsa-mir-100 mi-100 ZIP H Precursor Yes nd nahsa-miR-19b-3p mi-19b ZIP I Mature No nd No [39 40]hsa-mir-19b-1 mi-19b ZIP K Precursor Yes nd nahsa-miR-139-5p mi-139 ZIP L Mature Yes nd Yes [40]hsa-mir-139 mi-139 ZIP M Precursor Yes nd nahsa-miR-29a-3p mi-29a ZIP N Mature No nd No [39 40]hsa-mir-29a mi-29a ZIP O Precursor Yes nd nahsa-miR-199b-5p mi-199b ZIP P Mature Yes Yes (314)hsa-mir-199b mi-199b ZIP Q Precursor Yes Yes (320)hsa-miR-99a-5p mi-99a ZIP R Mature Yes Yes (211)hsa-mir-99a mi-99a ZIP S Precursor Yes Yes (334)

Log

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ensit

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100

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ZIP

Am

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9aZI

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9aZI

PO

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99b

ZIP

Pm

i-199

bZI

PQ

mi-9

9aZI

PR

mi-9

9aZI

PS

Figure 7 Detection of mature and precursor amiR in HeLa cellsSignal intensities from three hybridizations of fluorescently labeledMSO-cZIPs with the ZIP-code array after specific labeling arepresented Mature amiR was labeled with Cy3-dUTP and unlabeleddTTP using MSO-cZIP set A (gray) precursor amiR was labeledwith Cy3-dATP and unlabeled deoxynucleotides using MSO-cZIPset B (black) ZIPs for mature miRNA detection A C E G I L NP and R ZIPs for precursor miRNA detection B D F H KM O Qand S The red line indicates the defined threshold value (190 au)

The here studied miRNAs are located on separate pre-miRNA molecules If the assay is to be transferred to poly-cistronic miRNAs labeling of pre-miRs could be affected by

sterical hindrance through miR-primer binding in multiplexmiR labeling This would have to be clarified in furtherstudies

Based on synthetic templates we have proven the qual-ification of our method for the simultaneous detection anddistinction of mature and precursor miRNAs in principleand in order to further improve our method an optimizationof the MSO position could be pursued

Regarding the detection of miRNA in cells a furtheroptimization could also be performed by the establishmentof the ZIP-code system based on L-DNA analogue toHauser et al [35] with the aim of reducing backgroundsignals and preventing potential cross-hybridizations withD-DNA

In conclusion the comparative high-throughput-baseddetection of mature and precursor miRNA as tumor markersincluding variants has the potential for an improved tumorclassification This could allow an accurate diagnosis for apromising therapy especially in the case of very heteroge-neous tumor entities

Conflict of Interests

The authors have a patent pending on the method describedin this paper The authors do not have a direct financialrelation with the commercial entities mentioned in the paper


Acknowledgments

The authors are grateful to Professor Dr Fusenig (DKFZHeidelberg Germany) for providing HaCaT cell line and toIna Hogk and Doris Finkelmeier for cell culturing

References

[1] D P Bartel ldquoMicroRNAs genomics biogenesis mechanismand functionrdquo Cell vol 116 no 2 pp 281ndash297 2004

[2] N Bushati and S M Cohen ldquoMicroRNA functionsrdquo AnnualReview of Cell and Developmental Biology vol 23 pp 175ndash2052007

[3] G A Calin and C M Croce ldquoMicroRNA signatures in humancancersrdquo Nature Reviews Cancer vol 6 no 11 pp 857ndash8662006

[4] A Esquela-Kerscher and F J Slack ldquoOncomirsmdashmicroRNAswith a role in cancerrdquo Nature Reviews Cancer vol 6 no 4 pp259ndash269 2006

[5] E Barbarotto T D Schmittgen and G A Calin ldquoMicroRNAsand cancer profile profile profilerdquo International Journal ofCancer vol 122 no 5 pp 969ndash977 2008

[6] V N Kim and J W Nam ldquoGenomics of microRNArdquo Trends inGenetics vol 22 no 3 pp 165ndash173 2006

[7] J Yu F Wang G H Yang et al ldquoHuman microRNA clustersgenomic organization and expression profile in leukemia celllinesrdquo Biochemical and Biophysical Research Communicationsvol 349 no 1 pp 59ndash68 2006

[8] Y Lee M Kim J Han et al ldquoMicroRNA genes are transcribedby RNApolymerase IIrdquo EMBO Journal vol 23 no 20 pp 4051ndash4060 2004

[9] G M Borchert W Lanier and B L Davidson ldquoRNA poly-merase III transcribes human microRNAsrdquo Nature Structuraland Molecular Biology vol 13 no 12 pp 1097ndash1101 2006

[10] Y Lee C Ahn J Han et al ldquoThe nuclear RNase III Droshainitiates microRNA processingrdquo Nature vol 425 no 6956 pp415ndash419 2003

[11] J Han Y Lee K H Yeom et al ldquoMolecular basis for therecognition of primary microRNAs by the Drosha-DGCR8complexrdquo Cell vol 125 no 5 pp 887ndash901 2006

[12] E Lund S Guttinger A Calado J E Dahlberg and U KutayldquoNuclear export of microRNA precursorsrdquo Science vol 303 no5654 pp 95ndash98 2004

[13] E Bernstein A A Caudy S M Hammond and G J HannonldquoRole for a bidentate ribonuclease in the initiation step of RNAinterferencerdquo Nature vol 409 no 6818 pp 363ndash366 2001

[14] Y Kurihara and YWatanabe ldquoArabidopsis micro-RNA biogen-esis through Dicer-like 1 protein functionsrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 101 no 34 pp 12753ndash12758 2004

[15] K Forstemann Y Tomari T Du et al ldquoNormal microRNAmaturation and germ-line stem cell maintenance requiresLoquacious a double-stranded RNA-binding domain proteinrdquoPLoS Biology vol 3 no 7 article e236 2005

[16] G Meister and T Tuschl ldquoMechanisms of gene silencing bydouble-stranded RNArdquo Nature vol 431 no 7006 pp 343ndash3492004

[17] W Filipowicz S N Bhattacharyya and N Sonenberg ldquoMecha-nisms of post-transcriptional regulation by microRNAs are theanswers in sightrdquoNature Reviews Genetics vol 9 no 2 pp 102ndash114 2008

[18] T D Schmittgen ldquoRegulation of microRNA processing in de-velopment differentiation and cancerrdquo Journal of Cellular andMolecular Medicine vol 12 no 5 pp 1811ndash1819 2008

[19] V Ambros R C Lee A Lavanway P TWilliams andD JewellldquoMicroRNAs and other tiny endogenous RNAs in C elegansrdquoCurrent Biology vol 13 no 10 pp 807ndash818 2003

[20] J MThomsonM Newman J S Parker E MMorin-KensickiTWright and SMHammond ldquoExtensive post-transcriptionalregulation of microRNAs and its implications for cancerrdquoGenesand Development vol 20 no 16 pp 2202ndash2207 2006

[21] F GWulczyn L Smirnova A Rybak et al ldquoPost-transcription-al regulation of the let-7 microRNA during neural cell specifi-cationrdquo FASEB Journal vol 21 no 2 pp 415ndash426 2007

[22] N Yanaihara N Caplen E Bowman et al ldquoUnique microRNAmolecular profiles in lung cancer diagnosis and prognosisrdquoCancer Cell vol 9 no 3 pp 189ndash198 2006

[23] A J OrsquoHara W Vahrson and D P Dittmer ldquoGene alterationand precursor and mature microRNA transcription changescontribute to the miRNA signature of primary effusion lym-phomardquo Blood vol 111 no 4 pp 2347ndash2353 2008

[24] E S Jaffe ldquoThe 2008 WHO classification of lymphomasimplications for clinical practice and translational researchrdquoHematologythe Education Program of the American Society ofHematology pp 523ndash531 2009

[25] M Lagos-Quintana R Rauhut W Lendeckel and T TuschlldquoIdentification of novel genes coding for small expressedRNAsrdquoScience vol 294 no 5543 pp 853ndash858 2001

[26] A Valoczi C Hornyik N Varga J Burgyan S Kauppinenand Z Havelda ldquoSensitive and specific detection of microRNAsby northern blot analysis using LNA-modified oligonucleotideprobesrdquoNucleic Acids Research vol 32 no 22 article e175 2004

[27] E Varallyay J Burgyan and Z Havelda ldquoMicroRNA detectionby northern blotting using locked nucleic acid probesrdquo NatureProtocols vol 3 no 2 pp 190ndash196 2008

[28] T A Farazi J I Spitzer P Morozov and T Tuschl ldquoMiRNAs inhuman cancerrdquo Journal of Pathology vol 223 no 2 pp 102ndash1152011

[29] C Chen D A Ridzon A J Broomer et al ldquoReal-time quan-tification of microRNAs by stem-loop RT-PCRrdquo Nucleic AcidsResearch vol 33 no 20 article e179 2005

[30] T D Schmittgen J Jiang Q Liu and L Yang ldquoA high-throughput method to monitor the expression of microRNAprecursorsrdquo Nucleic Acids Research vol 32 no 4 article e432004

[31] W Li and K Ruan ldquoMicroRNA detection by microarrayrdquoAnalytical and Bioanalytical Chemistry vol 394 no 4 pp 1117ndash1124 2009

[32] C G Liu R Spizzo G A Calin and C M Croce ldquoExpressionprofiling of microRNA using oligo DNA arraysrdquo Methods vol44 no 1 pp 22ndash30 2008

[33] K A Cissell and S K Deo ldquoTrends in microRNA detectionrdquoAnalytical and Bioanalytical Chemistry vol 394 no 4 pp 1109ndash1116 2009

[34] M de Planell-Saguer and M C Rodicio ldquoAnalytical aspects ofmicroRNA in diagnostics a reviewrdquo Analytica Chimica Actavol 699 no 2 pp 134ndash152 2011

[35] N C Hauser R Martinez A Jacob S Rupp J D Hoheiseland S Matysiak ldquoUtilising the left-helical conformation of L-DNA for analysing different marker types on a single universalmicroarray platformrdquoNucleic Acids Research vol 34 no 18 pp5101ndash5111 2006


[36] M D Mattie C C Benz J Bowers et al ldquoOptimized high-throughput microRNA expression profiling provides novelbiomarker assessment of clinical prostate and breast cancerbiopsiesrdquoMolecular Cancer vol 5 article 24 2006

[37] QiagenMiScript PCR System Handbook 4th edition 2011[38] V Plaisance A Abderrahmani V Perret-Menoud P Jacque-

min F Lemaigre and R Regazzi ldquoMicroRNA-9 controls theexpression of GranuphilinSlp4 and the secretory response ofinsulin-producing cellsrdquo The Journal of Biological Chemistryvol 281 no 37 pp 26932ndash26942 2006

[39] P Landgraf M Rusu R Sheridan et al ldquoA mammalianmicroRNA expression atlas based on small RNA librarysequencingrdquo Cell vol 129 no 7 pp 1401ndash1414 2007

[40] A Lagana S Forte A Giudice et al ldquomiRo a miRNA knowl-edge baserdquo Database vol 2009 Article ID bap008 2009

[41] K Lemuth K Steuer and C Albermann ldquoEngineering ofa plasmid-free Escherichia coli strain for improved in vivobiosynthesis of astaxanthinrdquo Microbial Cell Factories vol 10article 29 2011

[42] S Griffiths-Jones H K Saini S van Dongen and A JEnright ldquomiRBase tools for microRNA genomicsrdquo NucleicAcids Research vol 36 no 1 pp D154ndashD158 2008

[43] J Starega-Roslan J Krol E Koscianska et al ldquoStructural basisof microRNA length varietyrdquoNucleic Acids Research vol 39 no1 pp 257ndash268 2011

[44] A Kozomara and S Griffiths-Jones ldquomiRBase integratingmicroRNA annotation and deep-sequencing datardquo NucleicAcids Research vol 39 no 1 pp D152ndashD157 2011


(miRNPs) and target specific mRNAs to trigger mRNAdegradation translational repression or both [6 16 17]

miRNA processing is regulated in development differ-entiation and cancer (reviewed in [18]) Changed levelsof mature miRNAs but unchanged levels of pri- and pre-miRNAs are the hallmark of regulated miRNA processingPosttranscriptional regulation ofmiRNAprocessing has beendescribed on the Drosha and Dicer levels [19ndash21]

Interestingly in tumor cells for example in lung cancer[3 22] or primary effusion lymphoma [23] an altered ratio ofprecursor and mature miRNA levels has been described Theprofiling of different miRNA types in parallel is important fora comprehensive cancer classification [23] especially in thecase of very heterogeneous cancersmdashlike lymphomas [24]Consequently differences in miRNA type levels have a highpotential as biomarkers and their detection might allow amore accurate characterization of different tumor subtypes

Currently precursor andmature miRNAs are detected byseveral molecularmethods (eg northern-blot [25ndash27] qRT-PCR [28ndash30] and DNA microarrays [3 31 32]) Northern-blot analysis allows the simultaneous detection of all miRNAtypes in parallel However the method is laborious andlimited in the number of analytes measured in parallel [33]qRT-PCR offers a higher sensitivity for detection but it alsoperforms on low-throughput level [34]

In contrast DNA microarrays are predestinated as ahigh-throughput method for miRNA detection in tumorsHowever commercially available array systems are not ableto detect the different miRNA types simultaneously with-out costly size exclusion (GeneChip miRNA 30 ArrayAffymetrix Santa Clara USA) or approximate data analysis(GenoExplorer microRNA system GenoSensor Corpora-tion Tempe USA) respectively because these array systemsonly detect the mature miRNA target sequence characteristicfor both miRNA types

Here we show aZIP-codeDNAmicroarray-based systemsimilar to Hauser et al [35] that allows the simultaneousdetection of precursor and mature miRNAs in one singleexperiment The array-platform setup uses unique and dis-tinct ZIP-code probes that show no cross-hybridization toany known organisms Specific primers attached to com-plementary ZIPs (cZIPs) are used for the specific labelingreactionThis allows the simultaneous detection of transcriptlevel variations genotypic differences and DNA-proteininteractions [35] We combined this array platform usingconventional DNA oligomers with a novel labeling approachto simultaneously detect precursor and mature miRNAs on aDNA microarray in one single experiment for a set of ninemiRNAs The approach has the potential for genome-widemiR analysis

2 Material and Methods

21 Cell Lines and Chemicals Human cervix carcinomacell line (HeLa) was obtained from the German Collectionof Microorganisms and Cell Cultures (Braunschweig Ger-many) Keratinocyte cell line (HaCaT) was kindly providedby Professor Dr Fusenig (DKFZ Heidelberg Germany)

All chemicals and reagents were obtained from Carl Roth(Karlsruhe Germany) or as indicated and they were of thehighest available purity

22 Cultivation of Human Cell Lines HeLa and HaCaT celllines were cultivated in 75 cm2 cell culture flasks (GreinerFrickenhausen Germany) and split 1 3 by standardmethodsjust before reaching confluence

All cell culture media were purchased from GIBCO(Life Technologies Darmstadt Germany) HeLa cells weremaintained in RPMI 1640 and HaCaT cells in DMEM (highglucose) each supplemented with 10 fetal calf serum (FCS)1 L-glutamine and 1 Penicillin-Streptomycin

All cells were cultivated under standard conditions at37∘C and 5 CO

2and passaged using Ca2+-Mg2+-free media

for HeLa cells and 10x trypsin-EDTA for HaCaT cells

23 Primers and Oligonucleotides All primers and oligonu-cleotides used in this work were purchased from Metabion(Planegg Germany) qPCR primers and T7 promoter oligo-nucleotides were ordered HPLC purified ZIP-code oligonu-cleotides synthetic oligonucleotides and miRNA specificoligonucleotide-cZIPs were ordered desalted

24 Distinct Labeling of Precursor and Mature miRNA

241 Preparation of Synthetic miRNA Templates Using thefollowing procedure synthetic miRNAs with a 51015840-polyA-tailwere prepared similar to a protocol for linear amplification ofmiRNAs published by Mattie et al [36]

A set of 9 miRNAs known to be involved in tumori-genesis was selected and corresponding mature and stem-loop sequences were extracted from miRBase database V18(httpwwwmirbaseorg correspondingmiRBase accessionnumbers see Table 1) Oligonucleotides reverse complemen-tary (rc) to the corresponding miRNA sequences with amaximum length of 80 nucleotides were designed containinga 14-mer poly d(T) sequence and the rc T7 RNA polymerasepromoter sequence at the 31015840-end (Table 1)

From thesemiRNADNAoligonucleotides T7-promoter-linked DNA oligonucleotides were produced therefore10 120583M of T7 promoter oligonucleotides (51015840-TAATACGAC-TCACTATAGGG-31015840) was mixed with 10 120583M of miRNADNA oligonucleotides and denatured for 3min at 95∘Cand strands were annealed at room temperature over nightThese T7-DNAs were used as an input template for in vitrotranscription (IVT)

Individual IVT reactions were performed for each T7-DNA all containing 5x transcription buffer (FermentasSt Leon-Rot Germany) 2mM NTPs (GE HealthcareMunchen Germany) 50U RNase inhibitor (RNase OutInvitrogen Darmstadt Germany) 30U T7 RNA polymerase(Fermentas St Leon-Rot Germany) and 1 120583g of the individ-ual T7-DNA IVT reactions were incubated for 14 hours at37∘C followed by purification of the amplified miRNA withRNeasy Min Elute Cleanup Kit (Qiagen Hilden Germany)according to the manufacturerrsquos instructions Quantificationof RNA amplificates was performed spectrophotometrically









































2(Invit-















1 2 3 4 5 6 7 8 9 10 11 12 13

(bp)

500400300

200

100

50











Length(nt)

Tm(∘C)

Ampliconlength (bp)

PCRefficiency

Errorrate ()







89 19 045



72 19 415














Mature miRNA

cZIP X

Cy3-dUTP +

dTTP

3998400

5998400

5998400

TTUTTTUTTTUTT

AAAAAAAAAAAAAA

(a)

Precursor miRNA

cZIP Y

Cy3-dATP+

dATPdCTPdGTPdTTP



3998400

5998400

5998400

GATGATTCCGA


(b)


miRNA(cZIP X)

ZIP Y

ZIP X

(c)













100

1000

10000

100000

Log

signa

l int

ensit

y (a

u)

mi-9

ZIP

Am

i-16

ZIP

Cm

i-92

ZIP

Em

i-100

ZIP

Gm

i-19b

ZIP

Im

i-139

ZIP

Lm

i-29a

ZIP

Nm

i-199

bZI

PP

mi-9

9aZI

PR

mi-9

ZIP

Bm

i-16

ZIP

Dm

i-92

ZIP

Fm

i-100

ZIP

Hm

i-19b

ZIP

Km

i-139

ZIP

Mm

i-29a

ZIP

Om

i-199

bZI

PQ

mi-9

9aZI

PS

(a)

100

1000

10000

100000

Log

signa

l int

ensit

y (a

u)

mi-9

ZIP

Am

i-16

ZIP

Cm

i-92

ZIP

Em

i-100

ZIP

Gm

i-19b

ZIP

Im

i-139

ZIP

Lm

i-29a

ZIP

Nm

i-199

bZI

PP

mi-9

9aZI

PR

mi-9

ZIP

Bm

i-16

ZIP

Dm

i-92

ZIP

Fm

i-100

ZIP

Hm

i-19b

ZIP

Km

i-139

ZIP

Mm

i-29a

ZIP

Om

i-199

bZI

PQ

mi-9

9aZI

PS

(b)





1

10

100

1000

10000

100000

Log

signa

l int

ensit

y (a

u)

mi-9

ZIP

Am

i-9ZI

PB

mi-1

6ZI

PC

mi-1

6ZI

PD

mi-9

2ZI

PE

mi-9

2ZI

PF

mi-1

00ZI

PG

mi-1

00ZI

PH

mi-1

9bZI

PI

mi-1

9bZI

PK

mi-1

39ZI

PL

mi-1

39ZI

PM

mi-2

9aZI

PN

mi-2

9aZI

PO

mi-1

99b

ZIP

Pm

i-199

bZI

PQ

mi-9

9aZI

PR

mi-9

9aZI

PS

(a)

Log

signa

l int

ensit

y (a

u)

1

10

100

1000

10000

100000

mi-9

ZIP

Am

i-9ZI

PB

mi-1

6ZI

PC

mi-1

6ZI

PD

mi-9

2ZI

PE

mi-9

2ZI

PF

mi-1

00ZI

PG

mi-1

00ZI

PH

mi-1

9bZI

PI

mi-1

9bZI

PK

mi-1

39ZI

PL

mi-1

39ZI

PM

mi-2

9aZI

PN

mi-2

9aZI

PO

mi-1

99b

ZIP

Pm

i-199

bZI

PQ

mi-9

9aZI

PR

mi-9

9aZI

PS

(b)






1

10

100

1000

10000

100000

Log

signa

l int

ensit

y (a

u)

mi-9

ZIP

Am

i-9ZI

PB

mi-1

6ZI

PC

mi-1

6ZI

PD

mi-9

2ZI

PE

mi-9

2ZI

PF

mi-1

00ZI

PG

mi-1

00ZI

PH

mi-1

9bZI

PI

mi-1

9bZI

PK

mi-1

39ZI

PL

mi-1

39ZI

PM

mi-2

9aZI

PN

mi-2

9aZI

PO

mi-1

99b

ZIP

Pm

i-199

bZI

PQ

mi-9

9aZI

PR

mi-9

9aZI

PS

(a)

1

10

100

1000

10000

100000

Log

signa

l int

ensit

y (a

u)

mi-9

ZIP

Am

i-9ZI

PB

mi-1

6ZI

PC

mi-1

6ZI

PD

mi-9

2ZI

PE

mi-9

2ZI

PF

mi-1

00ZI

PG

mi-1

00ZI

PH

mi-1

9bZI

PI

mi-1

9bZI

PK

mi-1

39ZI

PL

mi-1

39ZI

PM

mi-2

9aZI

PN

mi-2

9aZI

PO

mi-1

99b

ZIP

Pm

i-199

bZI

PQ

mi-9

9aZI

PR

mi-9

9aZI

PS

(b)













Log

signa

l int

ensit

y (a

u)

1

10

100

1000

10000

100000

mi-9

ZIP

A

mi-9

ZIP

B

mi-1

6ZI

PC

mi-1

6ZI

PD

mi-9

2ZI

PE

mi-9

2ZI

PF

mi-1

00ZI

PG

mi-1

00ZI

PH

mi-1

9bZI

PI

mi-1

9bZI

PK

mi-1

39ZI

PL

mi-1

39ZI

PM

mi-2

9aZI

PN

mi-2

9aZI

PO

mi-1

99b

ZIP

P

mi-1

99b

ZIP

Q

mi-9

9aZI

PR

mi-9

9aZI

PS

03 pmoles003 pmoles

0003 pmoles3 pmoles

(a)

Log

signa

l int

ensit

y (a

u)

1

10

100

1000

10000

100000

03 pmoles003 pmoles

0003 pmoles3 pmoles

mi-9

ZIP

A

mi-9

ZIP

B

mi-1

6ZI

PC

mi-1

6ZI

PD

mi-9

2ZI

PE

mi-9

2ZI

PF

mi-1

00ZI

PG

mi-1

00ZI

PH

mi-1

9bZI

PI

mi-1

9bZI

PK

mi-1

39ZI

PL

mi-1

39ZI

PM

mi-2

9aZI

PN

mi-2

9aZI

PO

mi-1

99b

ZIP

P

mi-1

99b

ZIP

Q

mi-9

9aZI

PR

mi-9

9aZI

PS

(b)












from triplicates)



Log

signa

l int

ensit

y (a

u)

100

1000

10000

mi-9

ZIP

Am

i-9ZI

PB

mi-1

6ZI

PC

mi-1

6ZI

PD

mi-9

2ZI

PE

mi-9

2ZI

PF

mi-1

00ZI

PG

mi-1

00ZI

PH

mi-1

9bZI

PI

mi-1

9bZI

PK

mi-1

39ZI

PL

mi-1

39ZI

PM

mi-2

9aZI

PN

mi-2

9aZI

PO

mi-1

99b

ZIP

Pm

i-199

bZI

PQ

mi-9

9aZI

PR

mi-9

9aZI

PS










Acknowledgments


References






















































































2(Invit-















1 2 3 4 5 6 7 8 9 10 11 12 13

(bp)

500400300

200

100

50











Length(nt)

Tm(∘C)

Ampliconlength (bp)

PCRefficiency

Errorrate ()







89 19 045



72 19 415














Mature miRNA

cZIP X

Cy3-dUTP +

dTTP

3998400

5998400

5998400

TTUTTTUTTTUTT

AAAAAAAAAAAAAA

(a)

Precursor miRNA

cZIP Y

Cy3-dATP+

dATPdCTPdGTPdTTP



3998400

5998400

5998400

GATGATTCCGA


(b)


miRNA(cZIP X)

ZIP Y

ZIP X

(c)













100

1000

10000

100000

Log

signa

l int

ensit

y (a

u)

mi-9

ZIP

Am

i-16

ZIP

Cm

i-92

ZIP

Em

i-100

ZIP

Gm

i-19b

ZIP

Im

i-139

ZIP

Lm

i-29a

ZIP

Nm

i-199

bZI

PP

mi-9

9aZI

PR

mi-9

ZIP

Bm

i-16

ZIP

Dm

i-92

ZIP

Fm

i-100

ZIP

Hm

i-19b

ZIP

Km

i-139

ZIP

Mm

i-29a

ZIP

Om

i-199

bZI

PQ

mi-9

9aZI

PS

(a)

100

1000

10000

100000

Log

signa

l int

ensit

y (a

u)

mi-9

ZIP

Am

i-16

ZIP

Cm

i-92

ZIP

Em

i-100

ZIP

Gm

i-19b

ZIP

Im

i-139

ZIP

Lm

i-29a

ZIP

Nm

i-199

bZI

PP

mi-9

9aZI

PR

mi-9

ZIP

Bm

i-16

ZIP

Dm

i-92

ZIP

Fm

i-100

ZIP

Hm

i-19b

ZIP

Km

i-139

ZIP

Mm

i-29a

ZIP

Om

i-199

bZI

PQ

mi-9

9aZI

PS

(b)





1

10

100

1000

10000

100000

Log

signa

l int

ensit

y (a

u)

mi-9

ZIP

Am

i-9ZI

PB

mi-1

6ZI

PC

mi-1

6ZI

PD

mi-9

2ZI

PE

mi-9

2ZI

PF

mi-1

00ZI

PG

mi-1

00ZI

PH

mi-1

9bZI

PI

mi-1

9bZI

PK

mi-1

39ZI

PL

mi-1

39ZI

PM

mi-2

9aZI

PN

mi-2

9aZI

PO

mi-1

99b

ZIP

Pm

i-199

bZI

PQ

mi-9

9aZI

PR

mi-9

9aZI

PS

(a)

Log

signa

l int

ensit

y (a

u)

1

10

100

1000

10000

100000

mi-9

ZIP

Am

i-9ZI

PB

mi-1

6ZI

PC

mi-1

6ZI

PD

mi-9

2ZI

PE

mi-9

2ZI

PF

mi-1

00ZI

PG

mi-1

00ZI

PH

mi-1

9bZI

PI

mi-1

9bZI

PK

mi-1

39ZI

PL

mi-1

39ZI

PM

mi-2

9aZI

PN

mi-2

9aZI

PO

mi-1

99b

ZIP

Pm

i-199

bZI

PQ

mi-9

9aZI

PR

mi-9

9aZI

PS

(b)






1

10

100

1000

10000

100000

Log

signa

l int

ensit

y (a

u)

mi-9

ZIP

Am

i-9ZI

PB

mi-1

6ZI

PC

mi-1

6ZI

PD

mi-9

2ZI

PE

mi-9

2ZI

PF

mi-1

00ZI

PG

mi-1

00ZI

PH

mi-1

9bZI

PI

mi-1

9bZI

PK

mi-1

39ZI

PL

mi-1

39ZI

PM

mi-2

9aZI

PN

mi-2

9aZI

PO

mi-1

99b

ZIP

Pm

i-199

bZI

PQ

mi-9

9aZI

PR

mi-9

9aZI

PS

(a)

1

10

100

1000

10000

100000

Log

signa

l int

ensit

y (a

u)

mi-9

ZIP

Am

i-9ZI

PB

mi-1

6ZI

PC

mi-1

6ZI

PD

mi-9

2ZI

PE

mi-9

2ZI

PF

mi-1

00ZI

PG

mi-1

00ZI

PH

mi-1

9bZI

PI

mi-1

9bZI

PK

mi-1

39ZI

PL

mi-1

39ZI

PM

mi-2

9aZI

PN

mi-2

9aZI

PO

mi-1

99b

ZIP

Pm

i-199

bZI

PQ

mi-9

9aZI

PR

mi-9

9aZI

PS

(b)













Log

signa

l int

ensit

y (a

u)

1

10

100

1000

10000

100000

mi-9

ZIP

A

mi-9

ZIP

B

mi-1

6ZI

PC

mi-1

6ZI

PD

mi-9

2ZI

PE

mi-9

2ZI

PF

mi-1

00ZI

PG

mi-1

00ZI

PH

mi-1

9bZI

PI

mi-1

9bZI

PK

mi-1

39ZI

PL

mi-1

39ZI

PM

mi-2

9aZI

PN

mi-2

9aZI

PO

mi-1

99b

ZIP

P

mi-1

99b

ZIP

Q

mi-9

9aZI

PR

mi-9

9aZI

PS

03 pmoles003 pmoles

0003 pmoles3 pmoles

(a)

Log

signa

l int

ensit

y (a

u)

1

10

100

1000

10000

100000

03 pmoles003 pmoles

0003 pmoles3 pmoles

mi-9

ZIP

A

mi-9

ZIP

B

mi-1

6ZI

PC

mi-1

6ZI

PD

mi-9

2ZI

PE

mi-9

2ZI

PF

mi-1

00ZI

PG

mi-1

00ZI

PH

mi-1

9bZI

PI

mi-1

9bZI

PK

mi-1

39ZI

PL

mi-1

39ZI

PM

mi-2

9aZI

PN

mi-2

9aZI

PO

mi-1

99b

ZIP

P

mi-1

99b

ZIP

Q

mi-9

9aZI

PR

mi-9

9aZI

PS

(b)












from triplicates)



Log

signa

l int

ensit

y (a

u)

100

1000

10000

mi-9

ZIP

Am

i-9ZI

PB

mi-1

6ZI

PC

mi-1

6ZI

PD

mi-9

2ZI

PE

mi-9

2ZI

PF

mi-1

00ZI

PG

mi-1

00ZI

PH

mi-1

9bZI

PI

mi-1

9bZI

PK

mi-1

39ZI

PL

mi-1

39ZI

PM

mi-2

9aZI

PN

mi-2

9aZI

PO

mi-1

99b

ZIP

Pm

i-199

bZI

PQ

mi-9

9aZI

PR

mi-9

9aZI

PS










Acknowledgments


References
























































1 2 3 4 5 6 7 8 9 10 11 12 13

(bp)

500400300

200

100

50











Length(nt)

Tm(∘C)

Ampliconlength (bp)

PCRefficiency

Errorrate ()







89 19 045



72 19 415














Mature miRNA

cZIP X

Cy3-dUTP +

dTTP

3998400

5998400

5998400

TTUTTTUTTTUTT

AAAAAAAAAAAAAA

(a)

Precursor miRNA

cZIP Y

Cy3-dATP+

dATPdCTPdGTPdTTP



3998400

5998400

5998400

GATGATTCCGA


(b)


miRNA(cZIP X)

ZIP Y

ZIP X

(c)













100

1000

10000

100000

Log

signa

l int

ensit

y (a

u)

mi-9

ZIP

Am

i-16

ZIP

Cm

i-92

ZIP

Em

i-100

ZIP

Gm

i-19b

ZIP

Im

i-139

ZIP

Lm

i-29a

ZIP

Nm

i-199

bZI

PP

mi-9

9aZI

PR

mi-9

ZIP

Bm

i-16

ZIP

Dm

i-92

ZIP

Fm

i-100

ZIP

Hm

i-19b

ZIP

Km

i-139

ZIP

Mm

i-29a

ZIP

Om

i-199

bZI

PQ

mi-9

9aZI

PS

(a)

100

1000

10000

100000

Log

signa

l int

ensit

y (a

u)

mi-9

ZIP

Am

i-16

ZIP

Cm

i-92

ZIP

Em

i-100

ZIP

Gm

i-19b

ZIP

Im

i-139

ZIP

Lm

i-29a

ZIP

Nm

i-199

bZI

PP

mi-9

9aZI

PR

mi-9

ZIP

Bm

i-16

ZIP

Dm

i-92

ZIP

Fm

i-100

ZIP

Hm

i-19b

ZIP

Km

i-139

ZIP

Mm

i-29a

ZIP

Om

i-199

bZI

PQ

mi-9

9aZI

PS

(b)





1

10

100

1000

10000

100000

Log

signa

l int

ensit

y (a

u)

mi-9

ZIP

Am

i-9ZI

PB

mi-1

6ZI

PC

mi-1

6ZI

PD

mi-9

2ZI

PE

mi-9

2ZI

PF

mi-1

00ZI

PG

mi-1

00ZI

PH

mi-1

9bZI

PI

mi-1

9bZI

PK

mi-1

39ZI

PL

mi-1

39ZI

PM

mi-2

9aZI

PN

mi-2

9aZI

PO

mi-1

99b

ZIP

Pm

i-199

bZI

PQ

mi-9

9aZI

PR

mi-9

9aZI

PS

(a)

Log

signa

l int

ensit

y (a

u)

1

10

100

1000

10000

100000

mi-9

ZIP

Am

i-9ZI

PB

mi-1

6ZI

PC

mi-1

6ZI

PD

mi-9

2ZI

PE

mi-9

2ZI

PF

mi-1

00ZI

PG

mi-1

00ZI

PH

mi-1

9bZI

PI

mi-1

9bZI

PK

mi-1

39ZI

PL

mi-1

39ZI

PM

mi-2

9aZI

PN

mi-2

9aZI

PO

mi-1

99b

ZIP

Pm

i-199

bZI

PQ

mi-9

9aZI

PR

mi-9

9aZI

PS

(b)






1

10

100

1000

10000

100000

Log

signa

l int

ensit

y (a

u)

mi-9

ZIP

Am

i-9ZI

PB

mi-1

6ZI

PC

mi-1

6ZI

PD

mi-9

2ZI

PE

mi-9

2ZI

PF

mi-1

00ZI

PG

mi-1

00ZI

PH

mi-1

9bZI

PI

mi-1

9bZI

PK

mi-1

39ZI

PL

mi-1

39ZI

PM

mi-2

9aZI

PN

mi-2

9aZI

PO

mi-1

99b

ZIP

Pm

i-199

bZI

PQ

mi-9

9aZI

PR

mi-9

9aZI

PS

(a)

1

10

100

1000

10000

100000

Log

signa

l int

ensit

y (a

u)

mi-9

ZIP

Am

i-9ZI

PB

mi-1

6ZI

PC

mi-1

6ZI

PD

mi-9

2ZI

PE

mi-9

2ZI

PF

mi-1

00ZI

PG

mi-1

00ZI

PH

mi-1

9bZI

PI

mi-1

9bZI

PK

mi-1

39ZI

PL

mi-1

39ZI

PM

mi-2

9aZI

PN

mi-2

9aZI

PO

mi-1

99b

ZIP

Pm

i-199

bZI

PQ

mi-9

9aZI

PR

mi-9

9aZI

PS

(b)













Log

signa

l int

ensit

y (a

u)

1

10

100

1000

10000

100000

mi-9

ZIP

A

mi-9

ZIP

B

mi-1

6ZI

PC

mi-1

6ZI

PD

mi-9

2ZI

PE

mi-9

2ZI

PF

mi-1

00ZI

PG

mi-1

00ZI

PH

mi-1

9bZI

PI

mi-1

9bZI

PK

mi-1

39ZI

PL

mi-1

39ZI

PM

mi-2

9aZI

PN

mi-2

9aZI

PO

mi-1

99b

ZIP

P

mi-1

99b

ZIP

Q

mi-9

9aZI

PR

mi-9

9aZI

PS

03 pmoles003 pmoles

0003 pmoles3 pmoles

(a)

Log

signa

l int

ensit

y (a

u)

1

10

100

1000

10000

100000

03 pmoles003 pmoles

0003 pmoles3 pmoles

mi-9

ZIP

A

mi-9

ZIP

B

mi-1

6ZI

PC

mi-1

6ZI

PD

mi-9

2ZI

PE

mi-9

2ZI

PF

mi-1

00ZI

PG

mi-1

00ZI

PH

mi-1

9bZI

PI

mi-1

9bZI

PK

mi-1

39ZI

PL

mi-1

39ZI

PM

mi-2

9aZI

PN

mi-2

9aZI

PO

mi-1

99b

ZIP

P

mi-1

99b

ZIP

Q

mi-9

9aZI

PR

mi-9

9aZI

PS

(b)












from triplicates)



Log

signa

l int

ensit

y (a

u)

100

1000

10000

mi-9

ZIP

Am

i-9ZI

PB

mi-1

6ZI

PC

mi-1

6ZI

PD

mi-9

2ZI

PE

mi-9

2ZI

PF

mi-1

00ZI

PG

mi-1

00ZI

PH

mi-1

9bZI

PI

mi-1

9bZI

PK

mi-1

39ZI

PL

mi-1

39ZI

PM

mi-2

9aZI

PN

mi-2

9aZI

PO

mi-1

99b

ZIP

Pm

i-199

bZI

PQ

mi-9

9aZI

PR

mi-9

9aZI

PS










Acknowledgments


References

















































Length(nt)

Tm(∘C)

Ampliconlength (bp)

PCRefficiency

Errorrate ()







89 19 045



72 19 415














Mature miRNA

cZIP X

Cy3-dUTP +

dTTP

3998400

5998400

5998400

TTUTTTUTTTUTT

AAAAAAAAAAAAAA

(a)

Precursor miRNA

cZIP Y

Cy3-dATP+

dATPdCTPdGTPdTTP



3998400

5998400

5998400

GATGATTCCGA


(b)


miRNA(cZIP X)

ZIP Y

ZIP X

(c)













100

1000

10000

100000

Log

signa

l int

ensit

y (a

u)

mi-9

ZIP

Am

i-16

ZIP

Cm

i-92

ZIP

Em

i-100

ZIP

Gm

i-19b

ZIP

Im

i-139

ZIP

Lm

i-29a

ZIP

Nm

i-199

bZI

PP

mi-9

9aZI

PR

mi-9

ZIP

Bm

i-16

ZIP

Dm

i-92

ZIP

Fm

i-100

ZIP

Hm

i-19b

ZIP

Km

i-139

ZIP

Mm

i-29a

ZIP

Om

i-199

bZI

PQ

mi-9

9aZI

PS

(a)

100

1000

10000

100000

Log

signa

l int

ensit

y (a

u)

mi-9

ZIP

Am

i-16

ZIP

Cm

i-92

ZIP

Em

i-100

ZIP

Gm

i-19b

ZIP

Im

i-139

ZIP

Lm

i-29a

ZIP

Nm

i-199

bZI

PP

mi-9

9aZI

PR

mi-9

ZIP

Bm

i-16

ZIP

Dm

i-92

ZIP

Fm

i-100

ZIP

Hm

i-19b

ZIP

Km

i-139

ZIP

Mm

i-29a

ZIP

Om

i-199

bZI

PQ

mi-9

9aZI

PS

(b)





1

10

100

1000

10000

100000

Log

signa

l int

ensit

y (a

u)

mi-9

ZIP

Am

i-9ZI

PB

mi-1

6ZI

PC

mi-1

6ZI

PD

mi-9

2ZI

PE

mi-9

2ZI

PF

mi-1

00ZI

PG

mi-1

00ZI

PH

mi-1

9bZI

PI

mi-1

9bZI

PK

mi-1

39ZI

PL

mi-1

39ZI

PM

mi-2

9aZI

PN

mi-2

9aZI

PO

mi-1

99b

ZIP

Pm

i-199

bZI

PQ

mi-9

9aZI

PR

mi-9

9aZI

PS

(a)

Log

signa

l int

ensit

y (a

u)

1

10

100

1000

10000

100000

mi-9

ZIP

Am

i-9ZI

PB

mi-1

6ZI

PC

mi-1

6ZI

PD

mi-9

2ZI

PE

mi-9

2ZI

PF

mi-1

00ZI

PG

mi-1

00ZI

PH

mi-1

9bZI

PI

mi-1

9bZI

PK

mi-1

39ZI

PL

mi-1

39ZI

PM

mi-2

9aZI

PN

mi-2

9aZI

PO

mi-1

99b

ZIP

Pm

i-199

bZI

PQ

mi-9

9aZI

PR

mi-9

9aZI

PS

(b)






1

10

100

1000

10000

100000

Log

signa

l int

ensit

y (a

u)

mi-9

ZIP

Am

i-9ZI

PB

mi-1

6ZI

PC

mi-1

6ZI

PD

mi-9

2ZI

PE

mi-9

2ZI

PF

mi-1

00ZI

PG

mi-1

00ZI

PH

mi-1

9bZI

PI

mi-1

9bZI

PK

mi-1

39ZI

PL

mi-1

39ZI

PM

mi-2

9aZI

PN

mi-2

9aZI

PO

mi-1

99b

ZIP

Pm

i-199

bZI

PQ

mi-9

9aZI

PR

mi-9

9aZI

PS

(a)

1

10

100

1000

10000

100000

Log

signa

l int

ensit

y (a

u)

mi-9

ZIP

Am

i-9ZI

PB

mi-1

6ZI

PC

mi-1

6ZI

PD

mi-9

2ZI

PE

mi-9

2ZI

PF

mi-1

00ZI

PG

mi-1

00ZI

PH

mi-1

9bZI

PI

mi-1

9bZI

PK

mi-1

39ZI

PL

mi-1

39ZI

PM

mi-2

9aZI

PN

mi-2

9aZI

PO

mi-1

99b

ZIP

Pm

i-199

bZI

PQ

mi-9

9aZI

PR

mi-9

9aZI

PS

(b)













Log

signa

l int

ensit

y (a

u)

1

10

100

1000

10000

100000

mi-9

ZIP

A

mi-9

ZIP

B

mi-1

6ZI

PC

mi-1

6ZI

PD

mi-9

2ZI

PE

mi-9

2ZI

PF

mi-1

00ZI

PG

mi-1

00ZI

PH

mi-1

9bZI

PI

mi-1

9bZI

PK

mi-1

39ZI

PL

mi-1

39ZI

PM

mi-2

9aZI

PN

mi-2

9aZI

PO

mi-1

99b

ZIP

P

mi-1

99b

ZIP

Q

mi-9

9aZI

PR

mi-9

9aZI

PS

03 pmoles003 pmoles

0003 pmoles3 pmoles

(a)

Log

signa

l int

ensit

y (a

u)

1

10

100

1000

10000

100000

03 pmoles003 pmoles

0003 pmoles3 pmoles

mi-9

ZIP

A

mi-9

ZIP

B

mi-1

6ZI

PC

mi-1

6ZI

PD

mi-9

2ZI

PE

mi-9

2ZI

PF

mi-1

00ZI

PG

mi-1

00ZI

PH

mi-1

9bZI

PI

mi-1

9bZI

PK

mi-1

39ZI

PL

mi-1

39ZI

PM

mi-2

9aZI

PN

mi-2

9aZI

PO

mi-1

99b

ZIP

P

mi-1

99b

ZIP

Q

mi-9

9aZI

PR

mi-9

9aZI

PS

(b)












from triplicates)



Log

signa

l int

ensit

y (a

u)

100

1000

10000

mi-9

ZIP

Am

i-9ZI

PB

mi-1

6ZI

PC

mi-1

6ZI

PD

mi-9

2ZI

PE

mi-9

2ZI

PF

mi-1

00ZI

PG

mi-1

00ZI

PH

mi-1

9bZI

PI

mi-1

9bZI

PK

mi-1

39ZI

PL

mi-1

39ZI

PM

mi-2

9aZI

PN

mi-2

9aZI

PO

mi-1

99b

ZIP

Pm

i-199

bZI

PQ

mi-9

9aZI

PR

mi-9

9aZI

PS










Acknowledgments


References
















































Mature miRNA

cZIP X

Cy3-dUTP +

dTTP

3998400

5998400

5998400

TTUTTTUTTTUTT

AAAAAAAAAAAAAA

(a)

Precursor miRNA

cZIP Y

Cy3-dATP+

dATPdCTPdGTPdTTP



3998400

5998400

5998400

GATGATTCCGA


(b)


miRNA(cZIP X)

ZIP Y

ZIP X

(c)













100

1000

10000

100000

Log

signa

l int

ensit

y (a

u)

mi-9

ZIP

Am

i-16

ZIP

Cm

i-92

ZIP

Em

i-100

ZIP

Gm

i-19b

ZIP

Im

i-139

ZIP

Lm

i-29a

ZIP

Nm

i-199

bZI

PP

mi-9

9aZI

PR

mi-9

ZIP

Bm

i-16

ZIP

Dm

i-92

ZIP

Fm

i-100

ZIP

Hm

i-19b

ZIP

Km

i-139

ZIP

Mm

i-29a

ZIP

Om

i-199

bZI

PQ

mi-9

9aZI

PS

(a)

100

1000

10000

100000

Log

signa

l int

ensit

y (a

u)

mi-9

ZIP

Am

i-16

ZIP

Cm

i-92

ZIP

Em

i-100

ZIP

Gm

i-19b

ZIP

Im

i-139

ZIP

Lm

i-29a

ZIP

Nm

i-199

bZI

PP

mi-9

9aZI

PR

mi-9

ZIP

Bm

i-16

ZIP

Dm

i-92

ZIP

Fm

i-100

ZIP

Hm

i-19b

ZIP

Km

i-139

ZIP

Mm

i-29a

ZIP

Om

i-199

bZI

PQ

mi-9

9aZI

PS

(b)





1

10

100

1000

10000

100000

Log

signa

l int

ensit

y (a

u)

mi-9

ZIP

Am

i-9ZI

PB

mi-1

6ZI

PC

mi-1

6ZI

PD

mi-9

2ZI

PE

mi-9

2ZI

PF

mi-1

00ZI

PG

mi-1

00ZI

PH

mi-1

9bZI

PI

mi-1

9bZI

PK

mi-1

39ZI

PL

mi-1

39ZI

PM

mi-2

9aZI

PN

mi-2

9aZI

PO

mi-1

99b

ZIP

Pm

i-199

bZI

PQ

mi-9

9aZI

PR

mi-9

9aZI

PS

(a)

Log

signa

l int

ensit

y (a

u)

1

10

100

1000

10000

100000

mi-9

ZIP

Am

i-9ZI

PB

mi-1

6ZI

PC

mi-1

6ZI

PD

mi-9

2ZI

PE

mi-9

2ZI

PF

mi-1

00ZI

PG

mi-1

00ZI

PH

mi-1

9bZI

PI

mi-1

9bZI

PK

mi-1

39ZI

PL

mi-1

39ZI

PM

mi-2

9aZI

PN

mi-2

9aZI

PO

mi-1

99b

ZIP

Pm

i-199

bZI

PQ

mi-9

9aZI

PR

mi-9

9aZI

PS

(b)






1

10

100

1000

10000

100000

Log

signa

l int

ensit

y (a

u)

mi-9

ZIP

Am

i-9ZI

PB

mi-1

6ZI

PC

mi-1

6ZI

PD

mi-9

2ZI

PE

mi-9

2ZI

PF

mi-1

00ZI

PG

mi-1

00ZI

PH

mi-1

9bZI

PI

mi-1

9bZI

PK

mi-1

39ZI

PL

mi-1

39ZI

PM

mi-2

9aZI

PN

mi-2

9aZI

PO

mi-1

99b

ZIP

Pm

i-199

bZI

PQ

mi-9

9aZI

PR

mi-9

9aZI

PS

(a)

1

10

100

1000

10000

100000

Log

signa

l int

ensit

y (a

u)

mi-9

ZIP

Am

i-9ZI

PB

mi-1

6ZI

PC

mi-1

6ZI

PD

mi-9

2ZI

PE

mi-9

2ZI

PF

mi-1

00ZI

PG

mi-1

00ZI

PH

mi-1

9bZI

PI

mi-1

9bZI

PK

mi-1

39ZI

PL

mi-1

39ZI

PM

mi-2

9aZI

PN

mi-2

9aZI

PO

mi-1

99b

ZIP

Pm

i-199

bZI

PQ

mi-9

9aZI

PR

mi-9

9aZI

PS

(b)













Log

signa

l int

ensit

y (a

u)

1

10

100

1000

10000

100000

mi-9

ZIP

A

mi-9

ZIP

B

mi-1

6ZI

PC

mi-1

6ZI

PD

mi-9

2ZI

PE

mi-9

2ZI

PF

mi-1

00ZI

PG

mi-1

00ZI

PH

mi-1

9bZI

PI

mi-1

9bZI

PK

mi-1

39ZI

PL

mi-1

39ZI

PM

mi-2

9aZI

PN

mi-2

9aZI

PO

mi-1

99b

ZIP

P

mi-1

99b

ZIP

Q

mi-9

9aZI

PR

mi-9

9aZI

PS

03 pmoles003 pmoles

0003 pmoles3 pmoles

(a)

Log

signa

l int

ensit

y (a

u)

1

10

100

1000

10000

100000

03 pmoles003 pmoles

0003 pmoles3 pmoles

mi-9

ZIP

A

mi-9

ZIP

B

mi-1

6ZI

PC

mi-1

6ZI

PD

mi-9

2ZI

PE

mi-9

2ZI

PF

mi-1

00ZI

PG

mi-1

00ZI

PH

mi-1

9bZI

PI

mi-1

9bZI

PK

mi-1

39ZI

PL

mi-1

39ZI

PM

mi-2

9aZI

PN

mi-2

9aZI

PO

mi-1

99b

ZIP

P

mi-1

99b

ZIP

Q

mi-9

9aZI

PR

mi-9

9aZI

PS

(b)












from triplicates)



Log

signa

l int

ensit

y (a

u)

100

1000

10000

mi-9

ZIP

Am

i-9ZI

PB

mi-1

6ZI

PC

mi-1

6ZI

PD

mi-9

2ZI

PE

mi-9

2ZI

PF

mi-1

00ZI

PG

mi-1

00ZI

PH

mi-1

9bZI

PI

mi-1

9bZI

PK

mi-1

39ZI

PL

mi-1

39ZI

PM

mi-2

9aZI

PN

mi-2

9aZI

PO

mi-1

99b

ZIP

Pm

i-199

bZI

PQ

mi-9

9aZI

PR

mi-9

9aZI

PS










Acknowledgments


References















































100

1000

10000

100000

Log

signa

l int

ensit

y (a

u)

mi-9

ZIP

Am

i-16

ZIP

Cm

i-92

ZIP

Em

i-100

ZIP

Gm

i-19b

ZIP

Im

i-139

ZIP

Lm

i-29a

ZIP

Nm

i-199

bZI

PP

mi-9

9aZI

PR

mi-9

ZIP

Bm

i-16

ZIP

Dm

i-92

ZIP

Fm

i-100

ZIP

Hm

i-19b

ZIP

Km

i-139

ZIP

Mm

i-29a

ZIP

Om

i-199

bZI

PQ

mi-9

9aZI

PS

(a)

100

1000

10000

100000

Log

signa

l int

ensit

y (a

u)

mi-9

ZIP

Am

i-16

ZIP

Cm

i-92

ZIP

Em

i-100

ZIP

Gm

i-19b

ZIP

Im

i-139

ZIP

Lm

i-29a

ZIP

Nm

i-199

bZI

PP

mi-9

9aZI

PR

mi-9

ZIP

Bm

i-16

ZIP

Dm

i-92

ZIP

Fm

i-100

ZIP

Hm

i-19b

ZIP

Km

i-139

ZIP

Mm

i-29a

ZIP

Om

i-199

bZI

PQ

mi-9

9aZI

PS

(b)





1

10

100

1000

10000

100000

Log

signa

l int

ensit

y (a

u)

mi-9

ZIP

Am

i-9ZI

PB

mi-1

6ZI

PC

mi-1

6ZI

PD

mi-9

2ZI

PE

mi-9

2ZI

PF

mi-1

00ZI

PG

mi-1

00ZI

PH

mi-1

9bZI

PI

mi-1

9bZI

PK

mi-1

39ZI

PL

mi-1

39ZI

PM

mi-2

9aZI

PN

mi-2

9aZI

PO

mi-1

99b

ZIP

Pm

i-199

bZI

PQ

mi-9

9aZI

PR

mi-9

9aZI

PS

(a)

Log

signa

l int

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(b)






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(a)

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Log

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1

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Log

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l int

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from triplicates)



Log

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y (a

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mi-9

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mi-1

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Acknowledgments


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Log

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Acknowledgments


References















































Log

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y (a

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Log

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Acknowledgments


References



















































from triplicates)



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l int

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Acknowledgments


References















































Acknowledgments


References














































Simultaneous Detection of Different MicroRNA Types Using ... file2 JournalofNucleicAcids (miRNPs)...

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Transcript of Simultaneous Detection of Different MicroRNA Types Using ... file2 JournalofNucleicAcids (miRNPs)...