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Bacterial Whole Genome Array Analysis

ses the sensitivity of array analysis by removing >95% of 16S and 23S rRNA from bacterial total RNA, leaving a highly enriched population of bacterial mRNA. The enriched mRNA serves as a superior template for synthesizing the labeled cDNA used in whole genome array analysis.


Pre-Isolation Factors That Affect RNA Quality
There are several key factors to consider before beginning any bacterial RNA isolation procedure. The first is when to harvest the bacteria to maximize yield and quality of intact RNA. The highest quality RNA is isolated from cells in the logarithmic phase of growth. RNA isolated from bacteria in the stationary phase typically exhibits increased degradation. For this reason, it is highly recommended that RNA not be harvested from cells that have grown into stationary phase unless a specific experiment requires such isolation.

A second factor to consider is the method used to harvest the bacteria. It is known that bacterial mRNAs exhibit a wide range of stabilities. For example, approximately 80% of all mRNAs in E. coli are known to have half-lives of between 3 and 8 minutes [2]. For this reason, it is important to collect cells in a manner that minimizes or eliminates the impact of harvesting conditions on both gene expression profiles and/or RNA quality. With this in mind, it is preferable to process small volumes of bacterial cultures (1-2 ml) and to use a brief centrifugation step (~1 min.) to pellet the cells. The cell pellets can then be processed with one of the following
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