Effect of Detergent Concentration and Type when printing on Epoxy Coated NEXTERION® Slide E

•

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This document summarizes the results of an experiment testing the effect of detergent concentration and type on signal intensity and spot morphology when printing oligonucleotides on NEXTERION® Slide E microarray slides. The experiment found that Triton X-100 at concentrations between 0.015-0.04% produced uniform spot intensity, shape, and diameter with low variability in spot sizes. Both detergent concentration and type influenced spot size and signal intensity, with lower concentrations producing less uniform spots and higher concentrations risking irregular spot morphology. The optimal detergent must be determined empirically based on the specific coating, probes, and printing method used.

Technology Business

Effect of Detergent Concentration and Type when
printing on Epoxy Coated NEXTERION® Slide E

29.07.2011

Experimental Details

§ Microarray Slide: NEXTERION® Slide E
§ Probe: 10 µM oligonucleotides, 70 mer in NEXTERION® Spot with varying
detergent types / detergent concentrations
§ Printing Method: Contact printing, SMP3 pins (Genetix QArray mini)
at 47.2 % rH; 22.1 °C
§ Immobilization: 30 min humidity chamber, 90% rH, RT; afterwards at ambient
§ Target: 10 nM complementary oligonucleotides, 24 mer
§ Hybridization: Automated hyb station (Tecan HS4800)
§ Scanning: 532 nm, 140 gain, 10 µm scan resolution (Tecan LS400 reloaded)
§ Data Analysis: Array-Pro Analyzer (Media Cybernetics, Inc.)

© SCHOTT Technical Glass Solutions GmbH Page 2

Signal Intensity – Detergent Concentration
NEXTERION® Spot - Signal Intensity vs. Detergent Concentration
50000
probe: 10µM oligo 70mer

45000

40000

35000

30000
Intensity [rfu]

25000

20000

15000

10000

5000

0
0.0025%

0.0025%

0.0025%

0.0025%
0.015%

0.005%

0.015%

0.005%

0.015%

0.005%

0.015%

0.005%
0.00125%

0.00125%

0.00125%

0.00125%
0.04%
0.03%
0.02%

0.01%

0.04%
0.03%
0.02%

0.01%

0.04%
0.03%
0.02%

0.01%

0.04%
0.03%
0.02%

0.01%
© SCHOTT Technical Glass Solutions GmbH Page 3
Tween 20 Triton X-100 Sarcosyl SDS

Spot Diameter – Detergent Concentration
NEXTERION® Spot - Spot Diameter vs. Detergent Concentration
350
probe: 10µM oligo 70mer

300

250
Spot Diameter [µm]

200

150

100

50

0
0.0025%

0.0025%

0.0025%

0.0025%
0.015%

0.005%

0.015%

0.005%

0.015%

0.005%

0.015%

0.005%
0.00125%

0.00125%

0.00125%

0.00125%
0.04%
0.03%
0.02%

0.01%

0.04%
0.03%
0.02%

0.01%

0.04%
0.03%
0.02%

0.01%

0.04%
0.03%
0.02%

0.01%
© SCHOTT Technical Glass Solutions GmbH
Tween 20 Triton X-100 Sarcosyl SDS Page 4

SDS – Spot Morphology
Diameter (mean) 280µm 285µm 269µm 244µm 204µm 165µm 146µm 98µm
Diameter (max) 309µm 375µm 343µm 313µm 287µm 260µm 241µm 143µm
Diameter (min) 254µm 250µm 242µm 218µm 173µm 112µm 80µm 66µm
Diameter (Diff. max-min) 55µm 125µm 101µm 95µm 114µm 148µm 161µm 77µm

0.04% 0.03% 0.02% 0.015% 0.01% 0.005% 0.0025% 0.00125%

© SCHOTT Technical Glass Solutions GmbH Page 5

Sarcosyl – Spot Morphology
Diameter (mean) 248µm 261µm 263µm 248µm 227µm 181µm 136µm 91µm
Diameter (max) 262µm 320µm 323µm 309µm 329µm 307µm 194µm 140µm
Diameter (min) 241µm 238µm 247µm 222µm 192µm 108µm 66µm 62µm
Diameter (Diff. max-min) 21µm 82µm 76µm 87µm 137µm 199µm 128µm 78µm

0.04% 0.03% 0.02% 0.015% 0.01% 0.005% 0.0025% 0.00125%

© SCHOTT Technical Glass Solutions GmbH Page 6

Triton X-100 – Spot Morphology
Diameter (mean) 126µm 124µm 127µm 123µm 114µm 105µm 104µm 95µm
Diameter (max) 142µm 140µm 144µm 143µm 155µm 185µm 170µm 144µm
Diameter (min) 109µm 107µm 116µm 112µm 97µm 77µm 66µm 61µm
Diameter (Diff. max-min) 33µm 33µm 28µm 31µm 58µm 108µm 104µm 83µm

0.04% 0.03% 0.02% 0.015% 0.01% 0.005% 0.0025% 0.00125%

© SCHOTT Technical Glass Solutions GmbH Page 7

Tween 20 – Spot Morphology
Diameter (mean) 95µm 96µm 122µm 139µm 121µm 111µm 112µm 100µm
Diameter (max) 106µm 142µm 169µm 187µm 151µm 169µm 200µm 158µm
Diameter (min) 84µm 80µm 92µm 108µm 101µm 88µm 80µm 68µm
Diameter (Diff. max-min) 22µm 62µm 77µm 79µm 50µm 81µm 120µm 90µm

0.04% 0.03% 0.02% 0.015% 0.01% 0.005% 0.0025% 0.00125%

© SCHOTT Technical Glass Solutions GmbH Page 8

Results and Conclusions

§ The best results in this test were obtained with:
- NEXTERION® Spot with Triton X-100
- Concentration range of 0.015% - 0.04%
- Uniform spot intensity / shape / diameter
- Lowest difference between minimal and maximal spot diameter (at good
spot morphology)
§ Spot size and signal intensity are influenced by the detergent concentration and
type used in NEXTERION® Spot print buffer.
§ Lower detergent concentrations generates non-uniform spot size.
§ With increased detergent concentration there is an increased risk of irregular spot
shapes / poor spot morphologies increases.
§ Optimal detergent type and concentration must be determined empirically, taking
into account; coating, probe type, probe concentration, immobilization and spotting
method.

© SCHOTT Technical Glass Solutions GmbH Page 9

Additional Information:
www.us.schott.com/nexterion

© SCHOTT Technical Glass Solutions GmbH Page 10

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Effect of Detergent Concentration and Type when printing on Epoxy Coated NEXTERION® Slide E

1. Effect of Detergent Concentration and Type when printing on Epoxy Coated NEXTERION® Slide E 29.07.2011

2. Experimental Details § Microarray Slide: NEXTERION® Slide E § Probe: 10 µM oligonucleotides, 70 mer in NEXTERION® Spot with varying detergent types / detergent concentrations § Printing Method: Contact printing, SMP3 pins (Genetix QArray mini) at 47.2 % rH; 22.1 °C § Immobilization: 30 min humidity chamber, 90% rH, RT; afterwards at ambient § Target: 10 nM complementary oligonucleotides, 24 mer § Hybridization: Automated hyb station (Tecan HS4800) § Scanning: 532 nm, 140 gain, 10 µm scan resolution (Tecan LS400 reloaded) § Data Analysis: Array-Pro Analyzer (Media Cybernetics, Inc.) © SCHOTT Technical Glass Solutions GmbH Page 2

3. Signal Intensity – Detergent Concentration NEXTERION® Spot - Signal Intensity vs. Detergent Concentration 50000 probe: 10µM oligo 70mer 45000 40000 35000 30000 Intensity [rfu] 25000 20000 15000 10000 5000 0 0.0025% 0.0025% 0.0025% 0.0025% 0.015% 0.005% 0.015% 0.005% 0.015% 0.005% 0.015% 0.005% 0.00125% 0.00125% 0.00125% 0.00125% 0.04% 0.03% 0.02% 0.01% 0.04% 0.03% 0.02% 0.01% 0.04% 0.03% 0.02% 0.01% 0.04% 0.03% 0.02% 0.01% © SCHOTT Technical Glass Solutions GmbH Page 3 Tween 20 Triton X-100 Sarcosyl SDS

4. Spot Diameter – Detergent Concentration NEXTERION® Spot - Spot Diameter vs. Detergent Concentration 350 probe: 10µM oligo 70mer 300 250 Spot Diameter [µm] 200 150 100 50 0 0.0025% 0.0025% 0.0025% 0.0025% 0.015% 0.005% 0.015% 0.005% 0.015% 0.005% 0.015% 0.005% 0.00125% 0.00125% 0.00125% 0.00125% 0.04% 0.03% 0.02% 0.01% 0.04% 0.03% 0.02% 0.01% 0.04% 0.03% 0.02% 0.01% 0.04% 0.03% 0.02% 0.01% © SCHOTT Technical Glass Solutions GmbH Tween 20 Triton X-100 Sarcosyl SDS Page 4

5. SDS – Spot Morphology Diameter (mean) 280µm 285µm 269µm 244µm 204µm 165µm 146µm 98µm Diameter (max) 309µm 375µm 343µm 313µm 287µm 260µm 241µm 143µm Diameter (min) 254µm 250µm 242µm 218µm 173µm 112µm 80µm 66µm Diameter (Diff. max-min) 55µm 125µm 101µm 95µm 114µm 148µm 161µm 77µm 0.04% 0.03% 0.02% 0.015% 0.01% 0.005% 0.0025% 0.00125% © SCHOTT Technical Glass Solutions GmbH Page 5

6. Sarcosyl – Spot Morphology Diameter (mean) 248µm 261µm 263µm 248µm 227µm 181µm 136µm 91µm Diameter (max) 262µm 320µm 323µm 309µm 329µm 307µm 194µm 140µm Diameter (min) 241µm 238µm 247µm 222µm 192µm 108µm 66µm 62µm Diameter (Diff. max-min) 21µm 82µm 76µm 87µm 137µm 199µm 128µm 78µm 0.04% 0.03% 0.02% 0.015% 0.01% 0.005% 0.0025% 0.00125% © SCHOTT Technical Glass Solutions GmbH Page 6

7. Triton X-100 – Spot Morphology Diameter (mean) 126µm 124µm 127µm 123µm 114µm 105µm 104µm 95µm Diameter (max) 142µm 140µm 144µm 143µm 155µm 185µm 170µm 144µm Diameter (min) 109µm 107µm 116µm 112µm 97µm 77µm 66µm 61µm Diameter (Diff. max-min) 33µm 33µm 28µm 31µm 58µm 108µm 104µm 83µm 0.04% 0.03% 0.02% 0.015% 0.01% 0.005% 0.0025% 0.00125% © SCHOTT Technical Glass Solutions GmbH Page 7

8. Tween 20 – Spot Morphology Diameter (mean) 95µm 96µm 122µm 139µm 121µm 111µm 112µm 100µm Diameter (max) 106µm 142µm 169µm 187µm 151µm 169µm 200µm 158µm Diameter (min) 84µm 80µm 92µm 108µm 101µm 88µm 80µm 68µm Diameter (Diff. max-min) 22µm 62µm 77µm 79µm 50µm 81µm 120µm 90µm 0.04% 0.03% 0.02% 0.015% 0.01% 0.005% 0.0025% 0.00125% © SCHOTT Technical Glass Solutions GmbH Page 8

9. Results and Conclusions § The best results in this test were obtained with: - NEXTERION® Spot with Triton X-100 - Concentration range of 0.015% - 0.04% - Uniform spot intensity / shape / diameter - Lowest difference between minimal and maximal spot diameter (at good spot morphology) § Spot size and signal intensity are influenced by the detergent concentration and type used in NEXTERION® Spot print buffer. § Lower detergent concentrations generates non-uniform spot size. § With increased detergent concentration there is an increased risk of irregular spot shapes / poor spot morphologies increases. § Optimal detergent type and concentration must be determined empirically, taking into account; coating, probe type, probe concentration, immobilization and spotting method. © SCHOTT Technical Glass Solutions GmbH Page 9

Effect of Detergent Concentration and Type when printing on Epoxy Coated NEXTERION® Slide E

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Effect of Detergent Concentration and Type when printing on Epoxy Coated NEXTERION® Slide E