Arfin S M, Long A D, Ito E T, Tolleri L, Riehle M M, Paegle E S, Hatfield G W
Department of Biological Chemistry, College of Medicine, University of California, Irvine, 92697, USA.
J Biol Chem. 2000 Sep 22;275(38):29672-84. doi: 10.1074/jbc.M002247200.
We have used nylon membranes spotted in duplicate with full-length polymerase chain reaction-generated products of each of the 4,290 predicted Escherichia coli K12 open reading frames (ORFs) to measure the gene expression profiles in otherwise isogenic integration host factor IHF(+) and IHF(-) strains. Our results demonstrate that random hexamer rather than 3' ORF-specific priming of cDNA probe synthesis is required for accurate measurement of gene expression levels in bacteria. This is explained by the fact that the currently available set of 4,290 unique 3' ORF-specific primers do not hybridize to each ORF with equal efficiency and by the fact that widely differing degradation rates (steady-state levels) are observed for the 25-base pair region of each message complementary to each ORF-specific primer. To evaluate the DNA microarray data reported here, we used a linear analysis of variance (ANOVA) model appropriate for our experimental design. These statistical methods allowed us to identify and appropriately correct for experimental variables that affect the reproducibility and accuracy of DNA microarray measurements and allowed us to determine the statistical significance of gene expression differences between our IHF(+) and IHF(-) strains. Our results demonstrate that small differences in gene expression levels can be accurately measured and that the significance of differential gene expression measurements cannot be assessed simply by the magnitude of the fold difference. Our statistical criteria, supported by excellent agreement between previously determined effects of IHF on gene expression and the results reported here, have allowed us to identify new genes regulated by IHF with a high degree of confidence.
我们使用了尼龙膜，这些膜上一式两份点样了由4290个预测的大肠杆菌K12开放阅读框（ORF）中的每一个经聚合酶链反应生成的全长产物，以测量在其他方面同基因的整合宿主因子IHF(+)和IHF(-)菌株中的基因表达谱。我们的结果表明，对于准确测量细菌中的基因表达水平，需要随机六聚体而非3' ORF特异性引物来合成cDNA探针。这可以通过以下事实来解释：目前可用的4290套独特的3' ORF特异性引物与每个ORF的杂交效率并不相同，以及对于与每个ORF特异性引物互补的每个信使RNA的25个碱基对区域，观察到了广泛不同的降解速率（稳态水平）。为了评估此处报告的DNA微阵列数据，我们使用了适合我们实验设计的线性方差分析（ANOVA）模型。这些统计方法使我们能够识别并适当校正影响DNA微阵列测量的可重复性和准确性的实验变量，并使我们能够确定我们的IHF(+)和IHF(-)菌株之间基因表达差异的统计显著性。我们的结果表明，可以准确测量基因表达水平的微小差异，并且不能简单地通过倍数差异的大小来评估差异基因表达测量的显著性。我们的统计标准，得到了先前确定的IHF对基因表达的影响与此处报告的结果之间的高度一致性的支持，使我们能够高度自信地鉴定受IHF调控的新基因。
