Immobilized probe and glass surface chemistry as variables in microarray fabrication.
BACKGROUND
Global gene expression studies with microarrays can offer biological insights never before possible. However, the technology possesses many sources of technical variability that are an obstacle to obtaining high quality data sets. Since spotted microarrays offer design/content flexibility and potential cost savings over commercial systems, we have developed prehybridization quality control strategies for spotted cDNA and oligonucleotide arrays. These approaches utilize a third fluorescent dye (fluorescein) to monitor key fabrication variables, such as print/spot morphology, DNA retention, and background arising from probe redistributed during blocking. Here, our labeled cDNA array platform is used to study, 1) compression of array data using known input ratios of Arabidopsis in vitro transcripts and arrayed serial dilutions of homologous probes; 2) how curing time of in-house poly-L-lysine coated slides impacts probe retention capacity; and 3) the retention characteristics of 13 commercially available surfaces.


RESULTS
When array element fluorescein intensity drops below 5,000 RFU/pixel, gene expression measurements become increasingly compressed, thereby validating this value as a prehybridization quality control threshold. We observe that the DNA retention capacity of in-house poly-L-lysine slides decreases rapidly over time (~50% reduction between 3 and 12 weeks post-coating; p < 0.0002) and that there are considerable differences in retention characteristics among commercially available poly-L-lysine and amino silane-coated slides.


CONCLUSIONS
High DNA retention rates are necessary for accurate gene expression measurements. Therefore, an understanding of the characteristics and optimization of protocols to an array surface are prerequisites to fabrication of high quality arrays.
Hessner
Martin J
MJ
The Max McGee National Research Center for Juvenile Diabetes, Department of Pediatrics, The Medical College of Wisconsin and Children's Hospital of Wisconsin, Milwaukee, WI 53226, USA. mhessner@mcw.edu
Meyer
Lisa
L
Tackes
Jennifer
J
Muheisen
Sanaa
S
Wang
Xujing
X
D000818:Animals; D017360:Arabidopsis / Q000235:genetics; D015342:DNA Probes / Q000737:chemistry*; D019793:Fluorescein; D005898:Glass; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation* / Q000379:methods / Q000592:standards; D011107:Polylysine; D051381:Rats; D015203:Reproducibility of Results; D013499:Surface Properties; D013997:Time Factors; D014466:Ultraviolet Rays
D015342:DNA Probes; D011107:Polylysine; D019793:Fluorescein