SCHOTT Nexterion has developed versatile, coated-patterned substrate architectures (MPX formats), which enable highly reproducible, multiplexed hybridization using a variety of probes types (PCR products, modified/unmodified oligos, antibodies, peptides, etc) and standard laboratory microarraying equipment.

1. Nexterion Slide MPX
Reproducible, Multiplexed Hybridization Using a Coated-Patterned Substrate Architecture
Heather Jakes, Joseph Granko, Jami DeLaney, Michael Wotring, Daniel Haines, Rajendra Redkar, and Samuel Conzone
SCHOTT Nexterion, A Division of SCHOTT North America, Inc., 400 York Avenue, Duryea, PA 18642
1 4 RESULTS 10
ABSTRACT Utilization of MPX platform provides exceptional sensitivity in DNA microarrays
Slide MPX allows researchers to work with commercial equipment Cy5 (Log) vs Cy3 (Log) signals indicating different expression 7
Although DNA microarray technology is widely used for global gene expression analysis, patterns in adjacent wells when hybridized with different target s
targets Detection Sensitivity
Significantly larger number of spots are detected
there is often a need to further investigate gene subsets involved in specific pathways, Similar patterns in wells 1 & 5 Similar patterns in wells 2 & 6 Similar patterns in wells 3 & 8 on MPX slides in comparison with non-MPX 100.00
toxicological responses, or for diagnostics purposes in a highly reproducible and extensive slides processed on the same day and/or 90.00
80.00
manner. SCHOTT Nexterion has developed versatile, coated-patterned substrate different day.
% Spots Found
70.00
10000- 1 00 0 0
10000- 100 00 10000- 1 00 0 0
60.00
architectures (MPX formats), which enable highly reproducible, multiplexed hybridization 50.00
Cy5 Intensity
Cy5 Intensity
Cy5 Intensity
F 5 3 2 M e d ia n - B 5 3 2
F 5 3 2 M e d ia n - B 5 3 2
40.00
using a variety of probes types (PCR products, modified/unmodified oligos, antibodies,
F 5 3 2 M e d ia n - B 5 3 2
30.00
peptides, etc) and standard laboratory microarraying equipment. A series of experiments 1000- 1 00 0 1000- 100 0 1000- 1 00 0
Note: Identical target amount and hybridization 20.00
volume was used in this comparison. 10.00
were performed on 16 well aminosilane coated-patterned substrates, which were printed 0.00
with 50-mer rat oligos and then simultaneously hybridized with multiple cDNA targets.
Nexterion Slide MPX 16 100- 1 00 100- 100
100- 1 00
MPX Non-MPX same day Non-MPX Diff day
SubstrateType
The multiplexed hybridization results were contrasted with those obtained using single- Well 1 Well 2 Well 3
10 10 1 |0 0 | 1 0 |0 0 0
10 | 1 0 |0 0 | |
1 0|0 0 |
10
10 100 1000
1 00 0
F 6 3 5 M e d ia n - B 6 3 5
10000
1 00 0 00
10
10
1 00 1 00 0 0 1 00 0 00
10
10 100 100 00 100 000
array assays in terms of reproducibility, sensitivity, and cost. Increased reproducibility was 100 1000
F 6 3 5 M e d ia n - B 6 3 5
10000 100 1000
F 6 3 5 M e d ia n - B 6 3 5
10000
Cy3 Intensity Cy3 Intensity Cy3 Intensity
realized using the multiplexed hybridization format as CVs were reduced by 30-50% over Target amount and volume can be further reduced in MPX hybridization
hybridization
single-array assays. Further, there was no loss of sensitivity when moving from a single Cy3 Kidney + Cy5 Liver Cy3 Kidney + Cy5 Heart Cy3 Heart + Cy5 Liver
to a multiplexed hybridization assay, and well-to-well cross contamination was insignificant Cy3: 600 PMT Cy 3 Cy 5
Cy5: 730 PMT
(<1%). Finally, the experimental costs were substantially reduced when using the MPX Identical subarrays in separate wells were
format due to the reduced usage of substrates, targets and reagent volumes. hybridized with 5 and 7.5 pmol of target in 30, 3
No rm aliz ed Sig n al
10000- 1 00 0 0
10000- 1000 0 10000- 1 00 0 0
45, and 60 µL of volume using DW structure.
Cy5 Intensity
Cy5 Intensity
Cy5 Intensity
2
F 5 3 2 M e d ia n - B 5 3 2
F 5 3 2 M e d ia n - B 5 3 2
F 5 3 2 M e d ia n - B 5 3 2
MATERIALS & METHODS The data based on GAPDH gene indicates that
1000-
1000- 1 00 0
1000- 1000
1 00 0
target amount and the hybridization volume can 1
Printing: Rat oligonucleotide probes (50-mer) purchased from MWG Biotech were printed be further reduced without affecting signal 0
intensity.
in 3X SSC + 10% DMSO at 20 µM concentration in the wells of aminosilane coated MPX 100- 1 00
100- 100
100- 1 00
60 uL 45 uL 30 uL 60 uL 45 uL 30 uL
substrates using a GeneMachines OminiGrid Accent Printer. Following printing, the Well 5 Well 6 10
Well 8
5 pmol Tar g e t
7.5 pmol
10 10 10 | 1 00 | 1 00 0 | 1 00 0 0
printed surface was rehydrated and then snap-dried for 10 seconds on a heat block A Deep Well superstructure is mated to a MPX 16 substrate
| | | | | |
10
10
1 00 1 00 0 1 00 0 0 1 00 0 00 10
10
100 1000 1000 0 1000 00
10 100 1000
F 6 3 5 M e d ia n - B 6 3 5
10000
Note: 7.5 pmol/60 µL used per well routinely.
F 6 3 5 M e d ia n - B 6 3 5
100 1000 10000 100 1000
F 6 3 5 M e d ia n - B 6 3 5
10000
Cy3 Intensity
(85ºC). The DNA was immobilized using a UV crosslinker (Stratagene) at 800 mJ. Cy3 Intensity Cy3 Intensity
2 5 8 11
intra-
Excellent intra-slide reproducibility is achieved using MPX substrates
Cross contamination can be avoided by using superstructures Utilization of MPX platform provides substantial cost benefits
cDNA generation and labeling: Kidney and liver mRNA (Stratagene) samples were Image of identical subarrays Pair-wise comparison between 8 subarrays
labeled using CyScribe cDNA Post Labeling Kit (Amersham). An average yield of 80-100 W ell 1 2
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3
0 .9 3
4
0 .9 3
5
0 .9 1
6
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7
0 .9 1
8
0 .9 3
Non-MPX MPX
1
pmol with specific activity of 30-50 (nucleotides/ dye molecule) for each dye was Image after multiplexed 1
Distinct targets in each well 2 0 .9 6
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0 .9 9 Material and reagent cost for processing a 16- 0.9
3
routinely obtained from 1 µg of mRNA. Equal amounts of Cy3 and Cy5 label (7.5 pmol
Normalized cost
hybridization 4 0 .9 3 0 .9 5 0 .9 8 0 .9 8 0 .9 8 0 .9 8 0 .9 9
well MPX substrate was compared with 16
0.8
0.7
each) were used per well in 45 µL (Low Volume or LV Structure) or 60 µL (Deep Well or 1 2 5
6
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0 .9 3
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