High reproducibility using sodium hydroxide-stripped long oligonucleotide DNA microarrays.
Recently, long oligonucleotide (60- to 70-mer) microarrays for two-color experiments have been developed and are gaining widespread use. In addition, when there is limited availability of mRNA from tissue sources, RNA amplification can and is being used to produce sufficient quantities of cRNA for microarray hybridization. Taking advantage of the selective degradation of RNA under alkaline conditions, we have developed a method to "strip" glass-based oligonucleotide microarrays that use fluorescent RNA in the hybridization, while leaving the DNA oligonucleotide probes intact and usable for a second experiment. Replicate microarray experiments conducted using stripped arrays showed high reproducibility, however, we found that arrays could only be stripped and reused once without compromising data quality. The intraclass correlation (ICC) between a virgin array and a stripped array hybridized with the same sample showed a range of 0.90-0.98, which is comparable to the ICC of two virgin arrays hybridized with the same sample. Using this method, once-stripped oligonucleotide microarrays are usable, reliable, and help to reduce costs.
Hu
Zhiyuan
Z
University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7295, USA.
Troester
Melissa
M
Perou
Charles M
CM
D001943:Breast Neoplasms / Q000235:genetics*; D045744:Cell Line, Tumor; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D015345:Oligonucleotide Probes / Q000737:chemistry* / Q000235:genetics*; D012313:RNA / Q000737:chemistry*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012972:Sodium Hydroxide / Q000737:chemistry*
D015345:Oligonucleotide Probes; D012972:Sodium Hydroxide; D012313:RNA