Efficient and reliable printing of high-density DNA microarrays with contact printers on standard...
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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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200
400
600
800
1000
1200
1400
1600
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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