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Effect of Detergent Concentration and Type when printing on Epoxy Coated NEXTERION® Slide E 29.07.2011
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Effect of Detergent Concentration and Type whenprinting on Epoxy Coated NEXTERION® Slide E
29.07.2011
Page 2© SCHOTT Technical Glass Solutions GmbH
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.)
Page 3© SCHOTT Technical Glass Solutions GmbH
Signal Intensity – Detergent ConcentrationNEXTERION® Spot - Signal Intensity vs. Detergent Concentration
0
5000
10000
15000
20000
25000
30000
35000
40000
45000
50000
0.04
%
0.03
%
0.02
%
0.01
5%
0.01
%
0.00
5%
0.00
25%
0.00
125%
0.04
%
0.03
%
0.02
%
0.01
5%
0.01
%
0.00
5%
0.00
25%
0.00
125%
0.04
%
0.03
%
0.02
%
0.01
5%
0.01
%
0.00
5%
0.00
25%
0.00
125%
0.04
%
0.03
%
0.02
%
0.01
5%
0.01
%
0.00
5%
0.00
25%
0.00
125%
Tween 20 Triton X-100 Sarcosyl SDS
Inte
nsity
[rfu
]
probe: 10µM oligo 70mer
Page 4© SCHOTT Technical Glass Solutions GmbH
Spot Diameter – Detergent ConcentrationNEXTERION® Spot - Spot Diameter vs. Detergent Concentration
0
50
100
150
200
250
300
350
0.04
%
0.03
%
0.02
%
0.01
5%
0.01
%
0.00
5%
0.00
25%
0.00
125%
0.04
%
0.03
%
0.02
%
0.01
5%
0.01
%
0.00
5%
0.00
25%
0.00
125%
0.04
%
0.03
%
0.02
%
0.01
5%
0.01
%
0.00
5%
0.00
25%
0.00
125%
0.04
%
0.03
%
0.02
%
0.01
5%
0.01
%
0.00
5%
0.00
25%
0.00
125%
Tween 20 Triton X-100 Sarcosyl SDS
Spot
Dia
met
er [µ
m]
probe: 10µM oligo 70mer
Page 5© SCHOTT Technical Glass Solutions GmbH
SDS – Spot Morphology
0.04% 0.03% 0.02% 0.015% 0.01% 0.005% 0.0025% 0.00125%
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
Page 6© SCHOTT Technical Glass Solutions GmbH
Sarcosyl – Spot Morphology
0.04% 0.03% 0.02% 0.015% 0.01% 0.005% 0.0025% 0.00125%
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
Page 7© SCHOTT Technical Glass Solutions GmbH
Triton X-100 – Spot Morphology
0.04% 0.03% 0.02% 0.015% 0.01% 0.005% 0.0025% 0.00125%
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
Page 8© SCHOTT Technical Glass Solutions GmbH
Tween 20 – Spot Morphology
0.04% 0.03% 0.02% 0.015% 0.01% 0.005% 0.0025% 0.00125%
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
Page 9© SCHOTT Technical Glass Solutions GmbH
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.
Page 10© SCHOTT Technical Glass Solutions GmbH
Additional Information:www.us.schott.com/nexterion
