Open AccessComparison of two probe preparation methods using long oligonucleotide microarrays
Author(s) -
Fahd Al-Mulla,
Raba’ Al-Tamimi,
Milad S. Bitar
Publication year - 2004
Publication title -
biotechniques/biotechniques
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.617
H-Index - 131
eISSN - 1940-9818
pISSN - 0736-6205
DOI - 10.2144/04375rr03
Subject(s) - oligonucleotide , dna microarray , computational biology , biology , dna , microbiology and biotechnology , genetics , chemistry , gene , gene expression
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.