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

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

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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 ...

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.

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

    • Experimental Details
      • Conclusions
      • 1. Nexterion ® Slide A+ is suited for high density arrays
      • 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
      • 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 ( h igh d ensity), 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 *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. 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 µm High 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 H 2 O 100% DMSO Pronto Universal Next spot HD Puffer B Evaporation of various print buffers
      • Very low evaporation of Next spot A HD
      Next spot HD Small and uniform spots 50% DMSO uniform spots, but donuts Pronto Universal biggest 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
      35K/55K 150/130  20 200 ++++ +++ 90/60 -- Next spot 35K/55K 150/130  7 0 600 ++++ ++++ 100/70 +++ Next spot HD 15K/25K 30K/35K 25K/35K Max. Array density (SMP3/SMP2) +++ +++ ++ Signal intensity ++ +++ ++++ Spot uniformity 600 200 600 Spots/filled pin 220/180 170/150 170/150 minimum pitch SMP3/SMP2  7 0  2 0  50 PreSpot (based on SMP3 pin) 120/90 120/90 120/90 Spot-Diameter SMP3/SMP2 +++ - ++ Evaporation Pronto Universal 3xSSC 50% DMSO With water -20°C Can be used additionally 80°C 2h Next spot Not necessary -20°C Can be used additionally 80°C or 6h 45°C followed by 2h 80°C 2h Next spot HD Vacuum dried or at – 20°C -20°C -20°C Storage of oligos Not necessary With water Not necessary Refilling of source plates after drying Can be used additionally Can be used additionally Can be used additionally UV crosslinking* 80°C 80°C 80°C Incubation temp. 2h 2h 2h Incubation Pronto Universal 3xSSC 50% DMSO Next spot HD Pitch 160µm 50% DMSO Pitch 220µm Pronto Universal Pitch 220µm Frequency distribution of spot diameter Next spot HD 50% DMSO Pronto Universal