Use of Microarrays with Different Probe Sizes for Monitoring GeneExpression
Microarrays with oligonucleotides of different lengths wereused to monitor gene expression at a whole-genome level. To determinewhat length of oligonucleotide is a better alternative to PCR-generatedprobes, the performance of oligonucleotide probes was systematicallycompared to that of their PCR-generated counterparts for 96 genes fromShewanella oneidensis MR-1 in terms of overall signal intensity, numbersof genes detected, specificity, sensitivity, and differential geneexpression under experimental conditions. Hybridizations conducted at42oC, 45oC, 50oC, and 60oC indicated that good sensitivities wereobtained at 45oC for oligonucleotide probes in the presence of 50 percentformamide, under which conditions specific signals were detected by bothPCR and oligonucleotide probes. Signal intensity increased as the lengthof the oligonucleotide probe increased, and the 70-mer oligonucleotideprobes produced signal intensities similar to the intensities obtainedwith the PCR probes and detected numbers of open reading frames similarto the numbers detected with the PCR probes. PCR amplicon, 70-mer,60-mer, and 50-mer arrays had detection sensitivities of 5.0, 25, 100,and 100 ng of genomic DNA, which were equivalent to approximately 1.9 x106, 9.2 x 106, 3.7 x 107, and 3.7 x 107 copies, respectively, when thearray was hybridized with genomic DNA. To evaluate differential geneexpression under experimental conditions, S. oneidensis MR-1 cells wereexposed to low- or high-pH conditions for 30 and 60 min, and thetranscriptional profiles detected by oligonucleotide probes (50-mer,60-mer, and 70-mer) were closely correlated with those detected by thePCR probes. The results demonstrated that 70-mer oligonucleotides canprovide the performance most comparable to the performance obtained withPCR-generated probes.
- Research Organization:
- COLLABORATION - OakRidge
- DOE Contract Number:
- DE-AC02-05CH11231
- OSTI ID:
- 903373
- Report Number(s):
- LBNL-60467; AEMIDF; R&D Project: VGTLJK; BnR: KP1102010; TRN: US200720%%305
- Journal Information:
- Applied and Environmental Microbiology, Vol. 71, Issue 9; Related Information: Journal Publication Date: Sept./2005; ISSN 0099-2240
- Country of Publication:
- United States
- Language:
- English
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