evaporation 10ul

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Conclusions

1. Nexterion® Slide A+ is suited for high density arrays

2. A few buffers are suited for high density arrays, whereas Next spot HD is the best

choice in our opinion as it allows arrays of 50K (SMP2 pins) and more

3. Next spot HD evaporates very low, which ensures uniform printing conditions over

the whole printing process

4. The post printing process for the according print buffers on Nexterion® slide A+

seems simple and fast

5. Pin sizes should be as small as possible (between 50 – 75µm tip diameter)

6. Other factors that affect the density of arrays are:

- hydrophobicity of surface

- printing humidity

- oligo concentration

- dwell time

Efficient and reliable printing of high-density DNA microarrays with contact printers on standard commercial slides

Oliver Kirchner, Matthias Schirmer, Christian Lautenschläger and Rüdiger Dietrich Schott Jenaer Glas GmbH Microarray Solutions Germany, 07745 Jena, Otto-Schott-Straße 13, +49 3641 91969

World Microarray Congress Vancouver 2007

Most uniform spots with Next spot HD on Nexterion® A+ slides

Slides Slide A+

Slide Processing Protocol for Nexterion® Slide A+

Probes rpoE (70-mer) with and without AminoLink dissolved in

Next spot HD (high density), 50% DMSO or Pronto Univ.

Spotting QArray Mini, SMP3/SMP2 pins

Target rpoE comp Dye 3 and Atto550

Hybridization Hybridization in Tecan HS4800

Scanning Tecan LS400 reloaded (488/532 nm)

Analysis ArrayPro Software

Experimental Details

50% 50% DMSODMSO Next spot HDNext spot HD Next spotNext spot 3xSSC3xSSC Pronto UniversalPronto Universal

EvaporationEvaporation ++ +++ -- - +++

Spot-Diameter Spot-Diameter SMP3/SMP2SMP3/SMP2

120/90 100/70 90/60 120/90 120/90

Spot uniformitySpot uniformity ++++ ++++ +++ +++ ++

Signal intensitySignal intensity ++ ++++ ++++ +++ +++

Spots/filled pinSpots/filled pin 600 600 200 200 600

PreSpot (based PreSpot (based on SMP3 pin)on SMP3 pin)

50 70 20 20 70

minimum pitch minimum pitch SMP3/SMP2SMP3/SMP2

170/150 150/130 150/130 170/150 220/180

Max. Array Max. Array density density

(SMP3/SMP2) (SMP3/SMP2) 25K/35K 35K/55K 35K/55K 30K/35K 15K/25K

*We recommend to do the baking step first, as the spots have to be dried before UV crosslinking anyway. In our hands the baking itself is sufficient.

50% DMSO50% DMSO Next spot HDNext spot HD Next spotNext spot 3xSSC3xSSC Pronto UniversalPronto Universal

IncubationIncubation 2h 2h 2h 2h 2h

Incubation temp.Incubation temp. 80°C80°C or 6h 45°C followed by 2h

80°C80°C 80°C 80°C

UV crosslinking*UV crosslinking*Can be used additionally

Can be used additionally

Can be used additionally

Can be used additionally

Can be used additionally

Storage of oligos Storage of oligos -20°C -20°C -20°C -20°CVacuum dried or at

–20°C

Refilling of source Refilling of source plates after drying plates after drying

Not necessary

Not necessary With water With water Not necessary

Next spot HDPitch 160µm

50% DMSOPitch 220µm

Pronto UniversalPitch 220µm

Signal intensity of various HD buffers on Nexterion® Slide A+ Spot morphology and uniformity of various HD buffers on Nexterion® Slide A+

Highest signal intensities with Next spot HD on Nexterion® A+

Minimum pitch of various HD buffers on Nexterion® Slide A+ (SMP2 pins)

High density print with Next spot HD on Nexterion® Slide A+

Pin tip diameter - 75µm (SMP3), pitch 160 µmHigh density print with Next spot HD on Nexterion® Slide A+

Pin tip diameter - 50µm (SMP2), pitch 130 µm

37K arrays printed with SMP3 pins and Next spot HD on Nexterion® A+ slides offer high reproducibility

50K arrays printed with SMP2 pins and Next spot HD on Nexterion® A+ slides offer high reproducibility

Abstract

Experimental details

Comparison of suitable print buffers for Nexterion® slide A+

Comparison of options for the post printing process for Nexterion® slide A+

Printing high density DNA microarrays is becoming more and more popular, and is invaluable for

"high-throughput" screening assays, and diagnostics applications such as aCGH. A higher density

array typically allows more assays to be performed simultaneously and/or, for lower sample

volumes to be used for the same number of assays. The ability to print microarrays in an efficient

and cost-effective manner, is of considerable interest to researchers.

A major issue with using contact printing is that the array elements should be consistently and

reliably positioned, and should be highly uniform. The array elements must be reproducible in size,

particularly for quantifying an analyte. In this study, some available printing buffers were tested for

their ability to print at high density over long print runs. The buffers were evaluated in terms of signal

yield, spot merging tendency (pitch), spot size and uniformity. In addition, the effect of pin size on

array density was analyzed on Aminosilane surfaces. It was demonstrated that with standard

contact printer and regular substrates, it is feasible to print 50K arrays that offer high reproducibility.

Hardware considerations (50K array with SMP2 pins)

I) Pins need to be perfectly adjusted, otherwise there could be problems in areas where two blocks of different pins are joining.

II) If printers work not precisely enough, slow motion modus might help to minimize this problem.

50% DMSO

H2O

100% DMSO

Pronto UniversalNext spot HD

Puffer B

Evaporation of various print buffers

Very low evaporation of Next spot A HD

Frequency distribution of spot diameter

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2200

80 85 90 95 100 105 110 115 120 125 130 135 140 145 150 155 160 165 170 175 180 185 190 195 200

Diameter [µm]

Am

ou

nt

GIW

DMSO

Pronto!U

Frequency distribution of spot diameter

Next spot HD

50% DMSO

Pronto Universal

Next spot HDSmall and uniform spots

50% DMSOuniform spots, but donuts

Pronto Universalbiggest spots and donuts

130 µm 110 µm

trouble free printing with 50

µm pins (tip diameter) recom-

mended at a pitch of 130µm

with Next spot HD