The reflective layers on Nexterion® HiSens significantly enhance sensitivity and signal response. The characteristics of the reflective layers have been optimized for the fluorescent wavelengths most commonly used in microarray experiments, and will simultaneously improve the performance in both the Cy3™ and Cy5™ channels. The slide is produced according to industry standard slide dimensions and is available with SCHOTT’s standard high quality functional coatings for DNA and protein microarraying. This means that the Nexterion® HiSens Slide is fully compatible with all microarray printing technologies and most slide processing protocols, allowing customers a smooth transition from industry-standard transparent microarray slides.
SCHOTT NEXTERION® HiSens optical coating for high sensitive microarray analysis
1. COMPANY PRESENTATION PRODUCT GROUP NEXTERION
SCHOTT Nexterion®
The Microarray Substrate Company
SCHOTT HiSens Slides:
A Cure for the Sensitivity
Blues – Achieve Higher
Signals with Less Target
2. COMPANY PRESENTATION PRODUCT GROUP NEXTERION
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
3. COMPANY PRESENTATION PRODUCT GROUP NEXTERION
SCHOTT’s solution is a fluorescence enhancing
slide coating
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
4. COMPANY PRESENTATION PRODUCT GROUP NEXTERION
How does the optical coating work?
1. Increase of excitation
Reflecting wave overlaps incoming wave Standing wave pattern
⇒
Organic
coating
Thin-film
dielectric
coating
Coating is optimized to give the maximum E-field field strength
in the vicinity of the surface ⇒ strongest fluorescence excitation
5. COMPANY PRESENTATION PRODUCT GROUP NEXTERION
How does the optical coating work?
2. Maximize reflection towards the detector
λex Fluorescence emission λex Fluorescence emission
Spots with labeled
DNA or oligos Organic
coating
Thin-film
dielectric
glass substrate
coating
(borofloat)
glass substrate
(borofloat)
within ~ 50 nm
fluorescence is enhanced
Coating is optimized to give the maximum reflection towards the detector
Both effects together ⇒ theoretically 12-fold
enhancement of slide performance
6. COMPANY PRESENTATION PRODUCT GROUP NEXTERION
Benchmark study on Nexterion® HiSens E
Which improvements can be obtained with
Nexterion® HiSens E slides?
7. COMPANY PRESENTATION PRODUCT GROUP NEXTERION
Objectives of the study
Comparing Slide E against Slide HiSens E against
the following parameters:
• Signal intensity
• Target concentrations used
• Number of genes detectable
• Data correlation
8. COMPANY PRESENTATION PRODUCT GROUP NEXTERION
Experimental Details
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)
9. COMPANY PRESENTATION PRODUCT GROUP NEXTERION
Higher Signals with Nexterion® HiSens E
AROSAROS Control
Oligos Oligos
Slide E Slide HiSens E
Intensity
Lo Hi
10. COMPANY PRESENTATION PRODUCT GROUP NEXTERION
Higher Signals with Nexterion® HiSens E
Comparison of Signal Intensity
Nexterion® Slide E vs. Slide HiSens E (Gain: 30 dB, Cy3)
Slide E
1 Slide HiSens E
0.9
0.8
0.7
Intensity
0.6
0.5
0.4
0.3
0.2
0.1
0 Background
1AA01
1AB01
1AC01
1AD01
1AE01
1AF01
1AG01
1AH01
1BA01
1BB01
1BC01
1BD01
1BE01
1BF01
1BG01
1BH01
Oligo/Gene
11. COMPANY PRESENTATION PRODUCT GROUP NEXTERION
More detectable signals with Nexterion® HiSens E
Comparison of Signal Intensity
Nexterion® Slide E vs. Slide HiSens E (Gain: 30 dB, Cy3)
Slide E
1 Slide HiSens E
0.9
0.8
0.7
Intensity
0.6
0.5
0.4
0.3
0.2
0.1
0 Background
1AA01
1AB01
1AC01
1AD01
1AE01
1AF01
1AG01
1AH01
1BA01
1BB01
1BC01
1BD01
1BE01
1BF01
1BG01
1BH01
25-35 % additional low expressor genes (30 of 96 testet on
this slide) are detectable with HiSens E only
Oligo/Gene
12. COMPANY PRESENTATION PRODUCT GROUP NEXTERION
8 fold higher performance with Nexterion® HiSensE
Signal Enhancement Factor
Nexterion ® Slide E vs. Slide HiSens E (Gain: 30 dB, Cy3)
17
16
15
14
13
12
11
10
Factor
9
8 Mean
7
6
5
4
3
2
1
0
1AA01
1AB01
1AC01
1AD01
1AE01
1AF01
1AG01
1AH01
1BA01
1BB01
1BC01
1BD01
1BE01
1BF01
1BG01
1BH01
Signals on average Oligo/Gene
amplified approx. 8-fold
13. COMPANY PRESENTATION PRODUCT GROUP NEXTERION
Ambion Inc.: Slide E versus HiSens E for miRNA arrays
miRNA fractions from lung and bladder were hybridized against printed oligos
20µg totalRNA equivalents on Slide E on HiSens E
14. COMPANY PRESENTATION PRODUCT GROUP NEXTERION
10 times less target and better signals!
20µg totalRNA equival. on E 2µg totalRNA equival. on HiSens E
15. COMPANY PRESENTATION PRODUCT GROUP NEXTERION
Quantification of signal-background
1. median signal – local background
2. normalization to ratios of slide E
20 µg. eq Sum Intensity
100000
90000
80000
70000
60000
RFU
50000
40000
30000
20000
10000
0
Oligo
avg E avg HiSens
• On average 7.8 fold increase of net signal
• 50% more signals detectable
16. COMPANY PRESENTATION PRODUCT GROUP NEXTERION
Correlation of Nexterion® HiSens E and Slide E data HiSens vs Slide E
Optimized Scanning
6 80000
Correlation
SlideE 20 µg & Slide E 5 µg = 0.991834
Slide E 20 µg & Slide E 2 µg = 0.958447 70000
4 Slide E 20 µg & HiSens 20 µg = 0.983246
Slide E 20 µg & HiSens 5 µg = 0.969035
Slide E 20 µg & HiSens 2 µg = 0.971587 60000
HiSens 20 µg & HiSens 5 µg = 0.990909
2 HiSens 20 µg & HiSens 2 µg = 0.973892
50000
Log(base2)R/G
0 40000
30000
-2
20000
-4
10000
-6 0
psID
SlideE 20ug avg SlideE 5ug avg SlideE 2 ug avg HiSens 20ug avg HiSens 5ug avg HiSens 2ug avg AVERAGE sum med. Int.
Ø High data correlation between slide E and HiSens E
Ø High data correlation between different target concentrations
17. COMPANY PRESENTATION PRODUCT GROUP NEXTERION
Dye swap experiments of Nexterion® HiSens E at
City of Hope National Medical Center
miRNA array with Ambion‘s mirVana probes dissolved in low evaporation buffer
Hu.Thymus 555 Hu.Thymus 555
Hu.Brain 647 Hu.Brain 647
18. COMPANY PRESENTATION PRODUCT GROUP NEXTERION
Data correlation
Hu.Thymus 555 Hu.Thymus 555
Hu.Brain 647 Hu.Brain 647
20000
hsa_mi
hsa_mir r_16
hsa_mir _26a 10000
hsa_mi ambi_mir hsa_mi
_124a r_205 hsa_mi
_7029 r_494
hsa_mir r_26a
_494
F532 Median - B532
F532 Median - B532
hsa_mir
2000
_16 1000
ambi_mir
_7029
hsa_mi
r_124a
hsa_mir
_205 100
200 2000 20000
200 2000 20000
F635 Median - B635 F635 Median - B635
Slide 31 Slide 30
Ø Data show excellent correlation with dye swap on HiSens E
19. COMPANY PRESENTATION PRODUCT GROUP NEXTERION
Different coatings and formats available....
Nexterion® Slide E Nexterion® HiSens
Epoxy chemistry for E, A and H chemistry
Nexterion® Slide A
Amino chemistry MPX format
for all functional coatings
Nexterion® Slide A+
Amino chemistry
MTP format
for E and A chemistry
Nexterion® Slide AL
Aldehyde chemistry
Nexterion® Slide H
NHS-ester chemistry
20. COMPANY PRESENTATION PRODUCT GROUP NEXTERION
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)
21. COMPANY PRESENTATION PRODUCT GROUP NEXTERION
SCHOTT Nexterion®
The Microarray Substrate Company
SCHOTT HiSens Slides:
A Cure for the Sensitivity
Blues – Achieve Higher
Signals with Less Target