Rosenow C, Saxena R M, Durst M, Gingeras T R
Affymetrix Inc., 3380 Central Expressway, Santa Clara, CA 95051, USA.
Nucleic Acids Res. 2001 Nov 15;29(22):E112. doi: 10.1093/nar/29.22.e112.
High density oligonucleotide arrays have been used extensively for expression studies of eukaryotic organisms. We have designed a prokaryotic high density oligonucleotide array using the complete Escherichia coli genome sequence to monitor expression levels of all genes and intergenic regions in the genome. Because previously described methods for preparing labeled target nucleic acids are not useful for prokaryotic cell analysis using such arrays, a mRNA enrichment and direct labeling protocol was developed together with a cDNA synthesis protocol. The reproducibility of each labeling method was determined using high density oligonucleotide probe arrays as a read-out methodology and the expression results from direct labeling were compared to the expression results from the cDNA synthesis. About 50% of all annotated E.coli open reading frames are observed to be transcribed, as measured by both protocols, when the cells were grown in rich LB medium. Each labeling method individually showed a high degree of concordance in replica experiments (95 and 99%, respectively), but when each sample preparation method was compared to the other, approximately 32% of the genes observed to be expressed were discordant. However, both labeling methods can detect the same relative gene expression changes when RNA from IPTG-induced cells was labeled and compared to RNA from uninduced E.coli cells.
高密度寡核苷酸阵列已被广泛用于真核生物的表达研究。我们利用完整的大肠杆菌基因组序列设计了一种原核生物高密度寡核苷酸阵列，以监测基因组中所有基因和基因间区域的表达水平。由于先前描述的制备标记靶核酸的方法不适用于使用此类阵列进行原核细胞分析，因此我们开发了一种mRNA富集和直接标记方案以及一种cDNA合成方案。使用高密度寡核苷酸探针阵列作为读出方法来确定每种标记方法的重现性，并将直接标记的表达结果与cDNA合成的表达结果进行比较。当细胞在丰富的LB培养基中生长时，通过这两种方案测量，约50%的所有注释的大肠杆菌开放阅读框被观察到有转录。在重复实验中，每种标记方法单独显示出高度的一致性（分别为95%和99%），但当将每种样品制备方法相互比较时，观察到表达的基因中约32%是不一致的。然而，当标记IPTG诱导细胞的RNA并与未诱导的大肠杆菌细胞的RNA进行比较时，两种标记方法都能检测到相同的相对基因表达变化。
