Use of microarrays with different probe sizes for monitoring gene expression.
Microarrays with oligonucleotides of different lengths were used to monitor gene expression at a whole-genome level. To determine what length of oligonucleotide is a better alternative to PCR-generated probes, the performance of oligonucleotide probes was systematically compared to that of their PCR-generated counterparts for 96 genes from Shewanella oneidensis MR-1 in terms of overall signal intensity, numbers of genes detected, specificity, sensitivity, and differential gene expression under experimental conditions. Hybridizations conducted at 42 degrees C, 45 degrees C, 50 degrees C, and 60 degrees C indicated that good sensitivities were obtained at 45 degrees C for oligonucleotide probes in the presence of 50% formamide, under which conditions specific signals were detected by both PCR and oligonucleotide probes. Signal intensity increased as the length of the oligonucleotide probe increased, and the 70-mer oligonucleotide probes produced signal intensities similar to the intensities obtained with the PCR probes and detected numbers of open reading frames similar to 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 x 10(6), 9.2 x 10(6), 3.7 x 10(7), and 3.7 x 10(7) copies, respectively, when the array was hybridized with genomic DNA. To evaluate differential gene expression under experimental conditions, S. oneidensis MR-1 cells were exposed to low- or high-pH conditions for 30 and 60 min, and the transcriptional profiles detected by oligonucleotide probes (50-mer, 60-mer, and 70-mer) were closely correlated with those detected by the PCR probes. The results demonstrated that 70-mer oligonucleotides can provide the performance most comparable to the performance obtained with PCR-generated probes.
He
Zhili
Z
Environmental Sciences Division, Oak Ridge National Laboratory, P.O. Box 2008, Oak Ridge, TN 37831-6038, USA.
Wu
Liyou
L
Fields
Matthew W
MW
Zhou
Jizhong
J
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D003470:Culture Media; D020869:Gene Expression Profiling; D015964:Gene Expression Regulation, Bacterial; D018869:Heat-Shock Response; D006863:Hydrogen-Ion Concentration; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D020592:Shewanella / Q000235:genetics / Q000254:growth & development / Q000378:metabolism / Q000502:physiology
D001426:Bacterial Proteins; D003470:Culture Media; D015345:Oligonucleotide Probes