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Many researchers still use aminosilane coated slides for microarray production by immobilizing the DNA probes on the surface via ionic interactions. However, epoxysilane coated slides, which allow to link probes covalently, becoming more and more accepted. The aim of this study was to compare both slides in their performance by using oligonucleotides (probe and target) as test system. We wanted to discover if there is a difference between both surfaces in terms of I) Binding capacity and signal intensity, II) Binding specificity, III) Background performance IV) Reproducibility. We used NEXTERION Slide E (epoxy) and NEXTERION Slide A+ (aminosilane) which are produced using identical production processes and analogous silanes. Therefore, these two slides types represent a suitable model to investigate the questions asked. In addition, epoxy and amine slides from other suppliers were included into the test. We discuss how these differences affect the degree of discrimination and specificity of microarray based experiments.
Citation preview
Conclusions
ü Epoxy slides display higher signal intensities and lower background producing higher signal to noise ratios than amine slidesü Epoxy slides have a higher DNA binding capacity than Amine slidesü Epoxy slides have a greater discrimination of mismatched oligos than amine slidesüEpoxy slides have a higher slide-to-slide reproducibility with correlation coefficients above 0.9.
üThis study indicates that for producing oligonucleotide microarrays, an Epoxy surface is ideally suited
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Slide ECorESlide A+UGAPS
Epoxy versus Amine Slides: Which is the better surface for immobilizing Oligonucleotides?Kornelia Kuhn, Markus Böhm, Katrin Steinmetzer
Schott Jenaer Glas GmbH Microarray Solutions Germany, 07745 Jena, Otto-Schott-Straße 13, +49 3641 91966
Experimental Procedures
Results Part VI - Reproducibility of Epoxy and Amine Slides
Amine and epoxy slides were used for gene expression analysis. Based on the pair wise comparison of signals intensities for each spot from two slides, the correlation coefficient* (R2) values were calculated to quantify the reproducibility of data within a slide type and among different slide types. Below the data of pair wise comparisons between two amine slides (left handed graph) and two epoxy slides (right handed graph) are shown as an example.
UGAPS 1 vs 2
R2 = 0.8977
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R2 = 0.9666
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Slide A Slide A+ UGAPS Slide E1vs2 0.781vs3 0.802vs3 0.90Average 0.83
SD
0.850.760.750.780.06
0.950.760.720.810.12 0.06
0.910.930.970.940.03
*R2 value of >0.9 indicates excellent correlation between 2 data sets.
ü The epoxy slide showed higher R2 values, indicating excellent repro-ducibility as compared to amine slides.
Results Part V - Representative Images of gene expression analysis
ü Background signals are lowest for epoxy slidesü Nonspecific binding appears more prevalent for amine slides.
Nexterion® Slide A Nexterion® Slide A+
Corning UltraGAPS Nexterion® Slide E
Oligonucleotide based test system for mechanistic studies
IconoClust (Clondiag Chip Technologies), signal-to-noise ratios are calculated by dividing the signal intensity by the standard deviation of the background signal
Analysis
Tecan LS400 reloadedScanning
Tecan Hybridization Station HS4800Hybridi-zation
Cy3-16merTarget
16mer, 48mer, 64mer (NH2-link) with one or two mismatches (MM1, MM2, respectively) or without mismatch (PM)
Probes
according to the manufacturers protocols, exception: tests of different blocking/deactivation protocols for Slide E and Slide A+, i.e. chemical blocking by Next Block (Slide E) or succinic anhydride (Slide A+) and deactivation using BSA (both, Slide E and A+)
Slide Processing
Spot III, Spot H50, 50% DMSOSpotting Solutions
QArray Mini, 22°C, 45% RH, SMP3 pin (Telechem),SpotterSchott Nexterion Slide E, A+, A, Corning Epoxy, U-GAPSSlides
Gene expression analysis using oligonucleotide probes and fluorescently labeled cDNA
GenePix 4.0 softwareAnalysisAxon 4000B scanner, 600 PMT for Cy3Scanning
Tecan Hybridization Station HS4800Hybridi-zation
Universal rat reference RNA (Stratagene, #740200) 15 µg per reaction/slide, Qiagen LabelStardirect labeling kit (Cy3-dCTP) and poly A-RNA from N. attenuata
Target
Operon Rat Oligo Set, version 1 (96 amino-modified oligos) at 20 µM + buffer control + 2 nonspecific DNA controls and N. attenuata probes (50mers)
Probes
according to the manufacturers protocolsSlide Processing
50% DMSO for aminosilane slides, Nexterion Spot for Slide ESpotting Solutions
Biorobotics Microgrid, 22°C, 45% RH,SpotterSCHOTT Nexterion Slides A, A+, E and Corning UltraGAPSSlides
Main differences between amine and epoxy slides
lower probability of non specific binding?
higher probability of non specific binding?
Influence on non-specific binding?
chemical inactivation (ringopening) → hydroxyl groups (very hydrophilic, not reactive) → lownon-specific binding
sterical hindrance (non-covalent adsorption of BSA) → protein layer with functional groups → nonspecific binding?orchemical inactivation (succinic anhydride) → carboxyl groups →non specific binding/modificationof exocyclic amine-groups and/or
Mechanism ofBlocking/inactivation
higher binding capacity?lower binding capacity?Influence on Binding Capacity?
by covalent binding (epoxy group reacts with amine and hydroxyl groups of DNA)
by adsorption and crosslinking →very complex and unpredictable
Mechanism of immobilization
Epoxy SlidesAmine Slides, poly-L-lysine Slides
Results Part III – Binding Specificity
5’-modified amine modified homologous oligonucleotides of different length carrying either no (perfect match – PM or two mismatches (MM 2) near the 3’-end and complementary Cy3-labeled oligonucelotides (16mers) where used to determine the specificity of immobilization and hybridization on epoxy and amine slides. A better discrimination was obtained with epoxy slides as compared to amine slides.We suppose that the DNA on amino slides is linked on more places compared to epoxy slides resulting in a lower specificty.
Results Part IV – Gene Expression Analysis
üIn general epoxy slides showed higher signal-to-noise ratios than amine slides.
üOnly amine slides showed false positive signals (marked in graph), whereas epoxy slides did not show false positive signals
üthe dynamic range of signal-to-noise ratios is larger for epoxy slides as compared to the ratios obtained with amine slides
Results Part I – Signal intensityAmine modified homologous oligonucleotides of different length (16mer, 48mer and 64mer) were immobilized and hybridized with Cy3-labeled 16mers.
false positive signals
AcknowledgementWe thank Dr. Rajendra Redkar (Schott North America) for kindly providing us with gene expression data from rat.We also thank Dr. Klaus Gase from the Max-Planck Institute for Chemical Ecology in Jena, Germany, for kindly providing us with probes sequences and total RNA from N. attenuata.
Similar comparisons were made for all slides tested, including amine slides (A, A+, Corning UltraGAPS (UGAPS) and Slide E (epoxy surface). The correlation coefficients are given in the table below.
5’-modified amine modified oligonucleotides were immobilized onto epoxy and amine slides. Poly-A RNA from N. attenuata was used to prepared fluorescently labeled cDNA (using aminoallyl-dUTP andNexterion Dye3).
Abstract
Most researchers use aminosilane coated slides for microarray production by immobilizing the DNA probes on the surface via ionic interactions. However, epoxysilane coated slides, which allow to link probes covalently, becoming more and more accepted. The aim of this study was to compare both slides in their performance by using oligonucleotides (probe and target) as test system. We wanted to know whether there is a difference between both surfaces in I) binding capacity and signal intensity, II) binding specificity, III) background performance and IV) reproducibility. We used Slide E (epoxy) and Slide A+ (amine) which are produced using identical production processes and analogous silanes. Therefore, these two slides types represent a suitable model to investigate the questions asked. In addition, epoxy and amine slides from other suppliers were included into the test. We discuss how these differences affect the degree of discrimination and specificity of microarray based experiments.
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Slide E
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Next E 64merNext E 48merNext E 16merNext A+ 64merNext A+ 48merNext A+ 16mer
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ü The epoxy slides (that use an active binding chemistry) have a higher binding capacity for oligos than amine slides (which rely on ionic interaction with subsequent crosslinking)
ü Significantly higher signal-to-noise ratios were obtained with theNexterion® epoxy slides than the amine slides (please note the logarithmic scale of y-axis).
Results Part II – Binding capacityAn amine modified oligonucleotide containing a Cy 3 label was immobilized and processed following the protocol (except addition of target)
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Epoxy (Nexterion® Slide E) Amine (Nexterion® Slide A+)
