Improved Print and QC Methods for Oligonucleotide Arrays

To determine if array spot quality asvisualized using the IDT QC reagent hasutility in predicting the quality of arrayperformance in real target hybridizations, wecompared the QC image obtained from aPCR product array with the image obtainedafter actual hybridization with labeledcellular RNAs (Figure 6). In mostinstances, the spot signal intensities from thetwo-color hybridization experiment mirrorthe spot signal intensities from the IDTCy3-nonamer QC hybridization. Notethat several spots are “missing” in bothimages. In several cases (see arrows), theQC reagent visualized probe spots that werenot seen in actual target RNA hybridization,presumably due to low representation ofthese specific RNA species in the target mix.Significantly, no cases were observed wherePCR product probe spots were detected inthe “real hybridization” and absent in theQC hybridization. Therefore the “missingspots” were successfully identified as either“print dropouts” or “target absence” usingthis QC method. The ability to discriminatebetween these two events is a critical goal ofarray QC analysis since it allows formeaningful interpretation of negativehybridization data. Thus, the IDTCy3-nonamer offers greater power forarray QC, with improved sensitivity andmore accurate assessment of spotted probequality as well as functional availability forhybridization.1-u1-a1-h1-A1-H7-u7-a7-hSigmaHybChecker(Cy5 replacement)[?µM]Molecular ProbesAlexa-546 Panomer(Cy3 )[7.5µM]GenetixSpotCheck(Cy3 )[10µM]TeleChemSeeIt(Cy3 )[10µM]IDT Nonamer(Cy3 )[2µM]Figure 4: Comparison of Nonamer QC Products. 70mer oligoprobes (1 is 25.7% GC and 7 is 55.7% GC) were printed @25µM using IDT’sOSB print buffer on UltraGAPS slides with a 3 hour humidity treatment andno heat coupling. Images are 5µm resolution scans using a ScanArray® 5000(Perkin Elmer) at 80/80 laser (power/gain) settings with the Cy5-channel forthe Sigma HybChecker and 73/73 laser (power/gain) settings with theCy3-channel for all others. Bold, black arrows indicate low GC-contentprobe-oligos immobilized on surface at low probe-density. (Pseudo-color scale:black