FIGURE 1. Comparison of the relative difference in transcript abundance detected by cDNA filter arrays and Northern blots.
Relative transcript abundance of several hypoxic genes (ERG11, HEM13, NCP1, and OLE1) and ACT1 (control) was quantified by both Northern blots and GeneFilter arrays (ResGen, Huntsville, AL). Changes in mRNA abundance were elicited by several treatments, including aerobic incubations in the presence or absence of 3 mM CoCl3 and anoxic incubations in the presence or absence of 80% CO. For all treatments, strain JM43 was grown in SSG-TEA medium to mid-exponential growth phase and RNA was isolated, hybridized, and quantified as described in the paper. The apparent difference in mRNA abundance relative to that of ACT1 mRNA is plotted as a function of the detection method used. A solid line of unity and a dashed regression line are shown. The figure shows that there is a good correlation (r2 = 0.94) between the relative differences in transcript abundance detected by gene-specific probes on Northern blots as compared to that detected by the filter arrays. The absolute difference in transcript abundance detected by the arrays is, however, predictably less (1.2-fold) than that detected by Northern blots.
FIGURE 2. Reproducibility of RNA labeling and hybridization.
A filter array was probed with 33P-labeled cDNA from strain JM43 grown aerobically in SSG-TEA medium, and the signal intensity of each ORF was quantified by phosphorimaging. The filter array was then stripped and probed with freshly prepared 33P-labeled cDNA that was generated from the same RNA sample. The hybridization intensity of each ORF is plotted as function of the two hybridizations. The figure shows that there is a good correlation (r2 = 0.98) between the relative intensities produced by the two hybridizations. For low abundance transcripts there was a slight decrease in raw intensity for the second labeling, as indicated by the deviation from the line of unity. The results show that the filter arrays can be stripped and reliably re-probed.
FIGURE 3. Cross-hybridization assessment.
A set of filter arrays was probed with 33P-labeled cDNA from strain JM43 grown aerobically in SSG-TEA medium. After a final room temperature wash in 0.5X SSC, 1% SDS for 20 min (ResGen's recommended protocol), the signal intensity of each ORF was quantified by phosphorimaging (24 h exposure). The same filter arrays were then washed at 42° C in 0.5X SSC, 1% SDS for 20 min and re-exposed to the phosphorimaging screen for 24 h. The raw hybridization intensity (arbitrary units) of each ORF is plotted as function of the final wash temperature. The largest decrease in hybridization intensity at the higher-temperature wash (>10-fold) occurred in ORFs that share an average sequence identity of 89% with one or more other ORFs in the genome. An examination of aerobic and hypoxic isoform gene pairs, respectively, AAC2/AAC3, COX5a/COX5b, CYC1/CYC7, and HYP2 (TIF51A)/ANB1, revealed positive hybridization results (raw intensity greater than or equal to 2 fold above mean background) for only one of the hypoxic isoform genes, ANB1, which shares 91% sequence identity to the aerobic gene HYP2. Although these results provide evidence of cross-hybridization, the raw hybridization intensity for ANB1 was substantially (7-fold) less than that of its aerobic counterpart HYP2. Increasing the final wash temperature to 55° C did not substantially lower the relative intensity ratio of ANB1 to HYP2 but did substantially decrease the total number of positive hybridization results Given that the 42° C final wash results in a high degree of hybridization specificity while preserving a high percentage of positive hybridization results, this washing procedure was used for all comparisons presented in the paper.

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