schott nexterion® hisens optical coating for high sensitive microarray analysis

COMPANY PRESENTATION PRODUCT GROUP NEXTERION

SCHOTT Nexterion®

The Microarray Substrate Company

SCHOTT HiSens Slides: A Cure for the SensitivityBlues – Achieve HigherSignals with Less Target


Approaches to increase signal or sensitivity

Obvious options

• Selection of appropriate slide surface• Optimized handling to achieve optimal signal/background ratios• Increase the dye load per target molecule

More sophisticated options

• Increase the dye load by for instance dendrimer technology or nanoparticles• Use of scanners with low intrinsic background levels• Use of optical coatings on slides


Nexterion® HiSens Slides

Ø Launched January 2006

Ø Innovative optical coating specifically

developed for microarray substrates

Ø Fully compatable with any attachement

chemistry and arraying hardware

Ø Unique coating developed using SCHOTT expertise in optical glass

technology

SCHOTT’s solution is a fluorescence enhancing slide coating


How does the optical coating work?1. Increase of excitation

⇒

Coating is optimized to give the maximum E-field field strength in the vicinity of the surface ⇒ strongest fluorescence excitation

Reflecting wave overlaps incoming wave

Thin-film dielectriccoating

Organiccoating

Standing wave pattern


2. Maximize reflection towards the detector

glass substrate(borofloat)

λex Fluorescence emission

Organiccoating

Thin-film dielectriccoatingglass substrate

(borofloat)

λex Fluorescence emission

Coating is optimized to give the maximum reflection towards the detector

How does the optical coating work?

Both effects together ⇒ theoretically 12-fold enhancement of slide performance

Spots with labeledDNA or oligos

within ~ 50 nm fluorescence is enhanced


Benchmark study on Nexterion® HiSens E

Which improvements can be obtained with

Nexterion® HiSens E slides?


Objectives of the study

• Signal intensity• Target concentrations used• Number of genes detectable• Data correlation

Comparing Slide E against Slide HiSens E against the following parameters:


Slides Nexterion® Slide E, Nexterion® HiSens E

Slide Processing standard protocols

Probes 70mer oligos, human AROS V4 (1 plate) + control plate

Spotting QArray Mini (Genetix), SMP3 pins (ArrayIt)

Target Dye 3 labeled cDNA derived from human brain/liver total RNA,

Dye 5 labeled cDNA derived from human liver/brain total RNA

Hybridization 2h - 8h, Tecan Hyb station HS4800

Scanning Affymetrix 428 and Tecan LS400 reloaded

Analysis IconoClust (Clondiag)

Experimental Details


Slide HiSens ESlide E

IntensityHiLo

AROSOligos

AROS ControlOligos

Higher Signals with Nexterion® HiSens E


Comparison of Signal IntensityNexterion vs. Slide (Gain: 30 dB, Cy3)® Slide E HiSens E

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nsity

Slide ESlide HiSens E

Higher Signals with Nexterion® HiSens E

Background


Comparison of Signal IntensityNexterion vs. Slide (Gain: 30 dB, Cy3)® Slide E HiSens E

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Slide ESlide HiSens E

More detectable signals with Nexterion® HiSens E

25-35 % additional low expressor genes (30 of 96 testet on this slide) are detectable with HiSens E only

Background


8 fold higher performance with Nexterion® HiSensE

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Signal Enhancement Factor Nexterion vs. (Gain: 30 dB, Cy3) ® Slide E Slide HiSens E

Mean

Signals on average amplified approx. 8-fold


Ambion Inc.: Slide E versus HiSens E for miRNA arrays

miRNA fractions from lung and bladder were hybridized against printed oligos20µg totalRNA equivalents on Slide E on HiSens E


10 times less target and better signals!

20µg totalRNA equival. on E 2µg totalRNA equival. on HiSens E


• On average 7.8 fold increase of net signal• 50% more signals detectable

Quantification of signal-background1. median signal – local background 2. normalization to ratios of slide E

20 µg. eq Sum Intensity

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avg E avg HiSens


HiSens vs Slide EOptimized Scanning

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SlideE 20ug avg SlideE 5ug avg SlideE 2 ug avg HiSens 20ug avg HiSens 5ug avg HiSens 2ug avg AVERAGE sum med. Int.

Correlation of Nexterion® HiSens E and Slide E data

Ø High data correlation between slide E and HiSens EØ High data correlation between different target concentrations

CorrelationSlideE 20 µg & Slide E 5 µg = 0.991834Slide E 20 µg & Slide E 2 µg = 0.958447Slide E 20 µg & HiSens 20 µg = 0.983246Slide E 20 µg & HiSens 5 µg = 0.969035Slide E 20 µg & HiSens 2 µg = 0.971587HiSens 20 µg & HiSens 5 µg = 0.990909HiSens 20 µg & HiSens 2 µg = 0.973892


Dye swap experiments of Nexterion® HiSens E at City of Hope National Medical Center

Hu.Thymus 555Hu.Brain 647


miRNA array with Ambion‘s mirVana probes dissolved in low evaporation buffer


Data correlation

F635 Median - B635200 2000 20000

F532

Med

ian

- B53

2

2000

20000

hsa_mir_205

hsa_mir_494

hsa_mir_124a

hsa_mir_26a

hsa_mir_16

ambi_mir_7029

F635 Median - B635200 2000 20000

F532

Med

ian

- B53

2

100

1000

10000 hsa_mir_205

hsa_mir_494

hsa_mir_124a

hsa_mir_26a

hsa_mir_16

ambi_mir_7029



Ø Data show excellent correlation with dye swap on HiSens E

Slide 31 Slide 30


Different coatings and formats available....

MPX formatfor all functional coatings

MTP formatfor E and A chemistry

Nexterion® Slide EEpoxy chemistry

Nexterion® Slide AAmino chemistry

Nexterion® Slide A+Amino chemistry

Nexterion® Slide ALAldehyde chemistry

Nexterion® Slide HNHS-ester chemistry

Nexterion® HiSensfor E, A and H chemistry


Conclusions

• On average 8 fold higher net signals

• 3-4 fold increased signal/noise ratio

• Identification of weakly expressed genes

25%-50% more genes detectable!

• Less target/probe required

• Protocols identical to conventional slides

• Compatible with all microarray equipment (except

scanners that image through the rear face of the slide)


SCHOTT Nexterion®

The Microarray Substrate Company

SCHOTT HiSens Slides: A Cure for the SensitivityBlues – Achieve HigherSignals with Less Target

schott nexterion® hisens optical coating for high sensitive microarray analysis

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