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.

1. 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
Abstract Results Part I – Signal intensity Results Part VI - Reproducibility of Epoxy and Amine
Results Part II – Binding capacity
Amine modified homologous oligonucleotides of different length (16mer,
An amine modified oligonucleotide containing a Cy 3 label was immobilized
Slides
Most researchers use aminosilane coated slides for microarray production by immobilizing the DNA probes 48mer and 64mer) were immobilized and hybridized with Cy3-labeled
on the surface via ionic interactions. However, epoxysilane coated slides, which allow to link probes and processed following the protocol (except addition of target)
16mers.
covalently, becoming more and more accepted. The aim of this study was to compare both slides in their Amine and epoxy slides were used for gene expression analysis. Based on the
performance by using oligonucleotides (probe and target) as test system. We wanted to know whether there pair wise comparison of signals intensities for each spot from two slides, the
is a difference between both surfaces in I) binding capacity and signal intensity, II) binding specificity, III) correlation coefficient* (R2) values were calculated to quantify the reproducibility
background performance and IV) reproducibility. We used Slide E (epoxy) and Slide A+ (amine) which are 100000
of data within a slide type and among different slide types.
Next E 64mer
produced using identical production processes and analogous silanes. Therefore, these two slides types Below the data of pair wise comparisons between two amine slides (left handed
Next E 48mer 14000
represent a suitable model to investigate the questions asked. In addition, epoxy and amine slides from Next E graph) and two epoxy slides (right handed graph) are shown as an example.
other suppliers were included into the test. We discuss how these differences affect the degree of Next E 16mer
discrimination and specificity of microarray based experiments. Next A+ 64mer 12000 Cor E
Next A+ 48mer Next A+ UGAPS 1 vs 2 Slide E 2 vs 3
Next A+ 16mer 10000 UGAPS
signal-to-noise
signal-to-noise
100000 100000
48mer, 64mer
R2 = 0.9666
R2 = 0.8977
8000
Main differences between amine and epoxy slides 10000
16mer
10000 10000
Slide 2
6000
Slide 3
1000 1000
Amine Slides, poly-L-lysine Slides Epoxy Slides 16mer
4000 100 100
48mer
Mechanism of immobilization by adsorption and crosslinking → by covalent binding (epoxy group 2000 10 10
64mer 10 100 1000 10000 100000 10 100 1000 10000 100000
very complex and unpredictable reacts with amine and hydroxyl Slide 1 Slide 2
groups of DNA) 1000 0
0 5 10 15 20 25 30 35 0 10 20 30 40 50
[probe] (µM)
Influence on Binding Capacity? lower binding capacity? higher binding capacity? [probe] (µM)
Similar comparisons were made for all slides tested, including amine slides (A,
A+, Corning UltraGAPS (UGAPS) and Slide E (epoxy surface). The correlation
ü The epoxy slides (that use an active binding chemistry) have a coefficients are given in the table below.
ü Significantly higher signal-to-noise ratios were obtained with the
Nexterion® epoxy slides than the amine slides (please note the higher binding capacity for oligos than amine slides (which rely on
Mechanism of sterical hindrance (non-covalent chemical inactivation (ring
logarithmic scale of y-axis). ionic interaction with subsequent crosslinking)
Blocking/inactivation adsorption of BSA) → protein layer opening) → hydroxyl groups (very Slide A Slide A+ UGAPS Slide E
with functional groups → non hydrophilic, not reactive) → low 1vs2 0.85 0.95 0.78 0.91
specific binding? non-specific binding 1vs3 0.76 0.76 0.80 0.93
or 2vs3 0.75 0.72 0.90 0.97
chemical inactivation (succinic Average 0.78 0.81 0.83 0.94
anhydride) → carboxyl groups → Epoxy (Nexterion® Slide E) SD 0.06 0.12 0.06 0.03
Amine (Nexterion® Slide A+)
non specific binding/modification Results Part III – Binding Specificity *R2 value of >0.9 indicates excellent correlation between 2 data sets.
of exocyclic amine-groups and/or 14000 14000
E MM2 E PM
Influence on non-specific binding? higher probability of non specific lower probability of non specific 12000 A+ MM2 UV A+ PM UV
12000
binding? binding? 5’-modified amine modified homologous oligonucleotides of different length carrying ü The epoxy slide showed higher R2 values, indicating excellent repro-
10000
either no (perfect match – PM or two mismatches (MM 2) near the 3’-end and 10000 ducibility as compared to amine slides.
signal-to-noise
complementary Cy3-labeled oligonucelotides (16mers) where used to determine the
signal-to-noise
8000 8000
specificity of immobilization and hybridization on epoxy and amine slides.
6000 6000
A better discrimination was obtained with epoxy slides as compared to
Experimental Procedures amine slides. 4000 4000
2000 2000
Conclusions
Oligonucleotide based test system for mechanistic studies We suppose that the DNA on amino slides is linked on more places compared to epoxy
slides resulting in a lower specificty. 0 0
Slides Schott Nexterion Slide E, A+, A, Corning Epoxy, U-GAPS 0 10 20 30 40 0 10 20 30 40 ü Epoxy slides display higher signal intensities and lower background
[probe] (µM) [probe] (µM)
Spotter QArray Mini, 22°C, 45% RH, SMP3 pin (Telechem), producing higher signal to noise ratios than amine slides
ü Epoxy slides have a higher DNA binding capacity than Amine slides
Spotting Spot III, Spot H50, 50% DMSO ü Epoxy slides have a greater discrimination of mismatched oligos than amine
Solutions
Results Part V - Representative Images of slides
Slide according to the manufacturers protocols, exception: tests of different blocking/deactivation
Processing protocols for Slide E and Slide A+, i.e. chemical blocking by Next Block (Slide E) or succinic
Results Part IV – Gene Expression Analysis gene expression analysis üEpoxy slides have a higher slide-to-slide reproducibility with correlation
anhydride (Slide A+) and deactivation using BSA (both, Slide E and A+) coefficients above 0.9.
Probes 16mer, 48mer, 64mer (NH2-link) with one or two mismatches (MM1, MM2, respectively) or
without mismatch (PM) 1000
Target Cy3-16mer Slide E
CorE
5’-modified amine modified
Hybridi- Tecan Hybridization Station HS4800 Slide A+
oligonucleotides were
signal to noise
zation 100 UGAPS
immobilized onto epoxy and Nexterion® Slide A Nexterion® Slide A+
Scanning Tecan LS400 reloaded amine slides. Poly-A RNA üThis study indicates that for producing oligonucleotide microarrays, an
from N. attenuata was used Epoxy surface is ideally suited
Analysis IconoClust (Clondiag Chip Technologies), signal-to-noise ratios are calculated by dividing the to prepared fluorescently
10
signal intensity by the standard deviation of the background signal labeled cDNA (using
aminoallyl-dUTP and
Nexterion Dye3).
Gene expression analysis using oligonucleotide probes and fluorescently labeled cDNA 1
Corning UltraGAPS Nexterion® Slide E
Acknowledgement
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We thank Dr. Rajendra Redkar (Schott North America) for kindly providing us with gene
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Spotter Biorobotics Microgrid, 22°C, 45% RH,
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expression data from rat.
probe
Spotting 50% DMSO for aminosilane slides, Nexterion Spot for Slide E Slide A+ We also thank Dr. Klaus Gase from the Max-Planck Institute for Chemical Ecology in Jena,
Solutions Germany, for kindly providing us with probes sequences and total RNA from N.
attenuata.
Slide according to the manufacturers protocols Cor E
false positive signals
Processing
Slide E
Probes Operon Rat Oligo Set, version 1 (96 amino-modified oligos) at 20 µM + buffer control + 2 üIn general epoxy slides showed higher signal-to-noise ratios than amine slides.
nonspecific DNA controls and N. attenuata probes (50mers) 0 100 200 300 400 500 600 700
rfu (gain 200 db)
Target Universal rat reference RNA (Stratagene, #740200) 15 µg per reaction/slide, Qiagen LabelStar üOnly amine slides showed false positive signals (marked in graph), whereas epoxy slides did not show
direct labeling kit (Cy3-dCTP) and poly A-RNA from N. attenuata false positive signals ü Background signals are lowest for epoxy slides
Hybridi- Tecan Hybridization Station HS4800
zation üthe dynamic range of signal-to-noise ratios is larger for epoxy slides as compared to the ratios ü Nonspecific binding appears more prevalent for
Scanning Axon 4000B scanner, 600 PMT for Cy3 obtained with amine slides amine slides.
Analysis GenePix 4.0 software