SCHOTT NEXTERION® HiSens optical coating for high sensitive microarray analysis
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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.
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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