Compatibility of DMSO spotting buffers with Nexterion Slide E Epoxy-coated Microarray slides

Final Report DMSO Spotting Buffers on Slide E Norman Rahnis, Kornelia Kuhn, Katrin Steinmetzer Application Support/R&D Microarray Solutions Schott Jenaer Glas GmbH

Experimental Details Spotting Solution Components DMSO, SSC, phosphate buffer, SDS Slides Nexterion Slide E Probes 70mer (from E. coli Genome Oligo Set Version 1.0) 10 µM Spotting QArray Mini, 22°C, 45% RH, SMP3 pin (Telechem), aQu Split Pin K2801 (diameter 150 µm) Target total RNA ( E.coli ) was used to synthesize aa-cDNA, the coupling of NHS-ester-dyes (Nexterion Dye3 and Dye5) was performed with a standard protocol derived from a Cy3/Cy5 labeling protocol, Hybridization Tecan Hybridization Station HS4800, in 5xSSC, 0.1% SDS, 0.1 mg/ml herring sperm DNA, 50% formamide, 42°C, 16 hrs Scanning Affymetrix Array Scanner 428 Analysis IconoClust

....some remarks
the following images show results after hybridization
blue spots mean lower signal, green spots mean higher signals
suitable buffers were highlighted in red
spot diameter was only determined for good spots

Results SMP3 Pin 50% DMSO 50% DMSO 3x SSC 50% DMSO 1.5x SSC, spot diameter 70 µm 50% DMSO 150 mM PP, 0.1% SDS 50% DMSO 150 mM PP, 0.05% SDS 50% DMSO 150 mM PP 50% DMSO

Results SMP3 Pin 30% DMSO 30% DMSO 3x SSC 30% DMSO 1.5x SSC, spot diameter 70 µm 30% DMSO 150 mM PP, 0.1% SDS 30% DMSO 150 mM PP, 0.05% SDS 30% DMSO 150 mM PP 30% DMSO

Results SMP3 Pin
best results were obtained when combining DMSO with SSC and with or without SDS (but only for f.c. SDS of 0.005%, higher concentrations lead to precipitation)
a combination of DMSO, phosphate buffer and SDS does not work
further tests with additional combinations of DMSO, SSC and other detergents will be tested
25% DMSO 0.005% SDS 50% DMSO 1.5x SSC, 0.005% SDS, spot diameter 60-70 µm 25% DMSO 1.5xSSC, 0.005% SDS Spot diameter 90 µm 50% DMSO 75 mM PP

Results SMP3 Pin
best results were obtained when combining DMSO with SSC and with or without detergents
spot sizes range between 100 and 140 µm
similar results were obtained with 30% DMSO
50% DMSO 2x SSC 50% DMSO 1.5x SSC, 0.025% CTAB 50% DMSO 1.5x SSC, 0.025% Triton X100 50% DMSO 2.5x SSC 50% DMSO 1.5x SSC, 0.005% SDS

Results aQu K2801 Pin 50% DMSO
no suitable buffer combinations
50% DMSO 3x SSC 50% DMSO 1.5x SSC 50% DMSO 150 mM PP, 0.1% SDS 50% DMSO 150 mM PP, 0.05% SDS 50% DMSO 150 mM PP 50% DMSO

Results aQu K2801 Pin 30% DMSO
30% DMSO with 1.5x SSC gives good results, further SSC-concentrations will be tested
30% DMSO 3x SSC spot diameter 360 µm 30% DMSO 1.5x SSC, spot diameter 90 µm 30% DMSO 150 mM PP, 0.1% SDS 30% DMSO 150 mM PP, 0.05% SDS 30% DMSO 150 mM PP 30% DMSO

Results aQu K2801 Pin
best results were obtained when combining DMSO with SSC and with or without SDS (but only for f.c. SDS of 0.005%, higher concentrations lead to precipitation)
a combination of DMSO, phosphate buffer and SDS does not work
further tests with additional combinations of DMSO, SSC and other detergents will be tested
25% DMSO 0.005% SDS 50% DMSO 1.5x SSC, 0.005% SDS, spot diameter 110 µm 25% DMSO 1.5xSSC, 0.005% SDS Spot diameter 160 µm 50% DMSO 75 mM PP

Results aQu K2801 Pin
best results were obtained when combining DMSO with SSC and with or without detergents
spot sizes range between 100 and 140 µm
similar results were obtained with 30% DMSO
50% DMSO 1.5x SSC, 0.025% CTAB 50% DMSO 1.5x SSC, 0.025% Triton X100 50% DMSO 2.5x SSC 50% DMSO 2x SSC 50% DMSO 1.5x SSC, 0.005% SDS

Summary
50 (30%) DMSO in water usually gives very small spots on Slide E (approximately 60 µm)
addition of SSC (f.c. 1.5x to 2.5x) with or without detergent (f.c. 0.005% to 0.025%) gives good spot morphology and size
spot size can be increased by increasing SSC concentration and/or adding detergent
50(30)% DMSO is not compatible with Next Spot or phosphate buffer (150 mM f.c. phosphate buffer), addition of detergent does not improve spot morphology*

Compatibility of DMSO spotting buffers with Nexterion Slide E Epoxy-coated Microarray slides

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Compatibility of DMSO spotting buffers with Nexterion Slide E Epoxy-coated Microarray slides Presentation Transcript