Comparison of two probe preparation methods using long oligonucleotide microarrays.
The use of oligonucleotides as a capture platform for microarray-based experiments is gaining popularity. Oligonucleotide-based microarrays involving various probe preparations have been compared by a number of researchers. Limited data are available, however, regarding the concordances and efficacies of various probe preparations on long oligonucleotide-based microarrays. Accordingly, the current investigation assesses two labeling methods, namely Atlas PowerScript fluorescent cDNA (random priming) and T7 in vitro transcription cRNA [poly(T) priming] labeling kits. Our data revealed that a high degree of reproducibility among the examined genes for each assay used with correlation coefficients of 0.93 and 0.94 for random priming and poly(T) priming, respectively. It is worthy of note, however, that when the two assaying methods were compared, the data showed a poor correlation coefficient. A confirmatory step involving real-time reverse transcription PCR (RT-PCR) of 18 selected genes favors the superiority of the cDNA fluorescent labeling over the T7 labeling method. Overall, the microarray results generated by the poly(T) priming methodology should be viewed cautiously even when high reproducibility is evident.
Al-Mulla
Fahd
F
Kuwait University, Faculty of Medicine, Department of Pathology, Safat, Kuwait. fahd@al-mulla.org
Al-Tamimi
Raba'
R
Bitar
Milad S
MS
D015342:DNA Probes / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry*; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013194:Staining and Labeling / Q000379:methods*
D015342:DNA Probes; D005456:Fluorescent Dyes