Analysis of the quality of contact-pin fabricated oligonucleotide microarrays.
As the quality of microarrays is critical to successful experiments for data consistency and validity, a reliable and convenient quality control method is needed. We describe a systematic quality control method for large-scale genome oligonucleotide arrays. This method is comprised of three steps to assess the quality of printed arrays. The first step involves assessment of the autofluorescence property of DNA. This step is convenient, quick to perform, and allowed reuse of every array. The second step involves hybridization of arrays with Cy3-labeled 9-mer oligonucleotide target to assess the quality and stability of oligonucleotides. Because this step consumed arrays, one or two arrays from each batch were used to complement the quality control data from autofluorescence. The third step involves hybridization of arrays from every batch with transcripts derived from two cell lines to assess data consistency. These hybridizations were able to distinguish two closely related tissue samples by identifying a cluster of 20 genes that were differently expressed in U87MG and T98G glioblastoma cell lines. In addition, we standardized two parameters that significantly enhanced the quality of arrays. We found that longer pin contact time and crosslinking oligonucleotides at 400 mJ/cm(2) were optimal for the highest hybridization intensity. Taken together, these results indicate that the quality of spotted oligonucleotide arrays should be assessed by at least two methods, autofluorescence and 9-mer hybridization before arrays are used for hybridization experiments.
Yang
Amy X
AX
Tumor Vaccines and Biotechnology Branch, Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research, Food and Drug Administration, RM2NN20, Building 29B, 8800 Rockville Pike, Bethesda, MD 20892, USA.
Mejido
Josef
J
Bhattacharya
Bhaskar
B
Petersen
David
D
Han
Jing
J
Kawasaki
Ernest S
ES
Puri
Raj K
RK
D000818:Animals; D001921:Brain / Q000473:pathology / Q000821:virology; D001923:Brain Chemistry; D002232:Carbocyanines / Q000032:analysis; D045744:Cell Line, Tumor / Q000737:chemistry; D018076:DNA, Complementary / Q000235:genetics; D004273:DNA, Neoplasm / Q000235:genetics; D004685:Encephalomyelitis, Venezuelan Equine / Q000473:pathology; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000032:analysis; D005470:Fluorometry; D020869:Gene Expression Profiling / Q000295:instrumentation*; D005909:Glioblastoma / Q000473:pathology; D006801:Humans; D051379:Mice; D009363:Neoplasm Proteins / Q000235:genetics; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation / Q000592:standards*; D015345:Oligonucleotide Probes / Q000528:radiation effects; D011107:Polylysine; D011786:Quality Control; D013382:Subtraction Technique; D013997:Time Factors; D014158:Transcription, Genetic; D014466:Ultraviolet Rays
D002232:Carbocyanines; D018076:DNA, Complementary; D004273:DNA, Neoplasm; D005456:Fluorescent Dyes; D009363:Neoplasm Proteins; D015345:Oligonucleotide Probes; C089187:cyanine dye 3; D011107:Polylysine