In previous chapters, we have discussed the formats, methods of manufacture, and data aquisition instruments required for gene expression profiling with DNA arrays. In this chapter, we consider issues such as: heterogeneities encountered among experimental samples, isolation procedures for non-polyadenylated and polyadenylated RNA from bacteria and higher organisms; advantages and disadvantages of different target preparation methods; and general problems encountered during the execution of such experiments. Throughout this chapter we focus on ways to minimize experimental errors. In particular, we point out the pitfalls of current methods for the preparation of targets from polyadenylated RNA and discuss alternative methods of target synthesis from total RNA preparations from eukaryotic cells based on methods developed for the synthesis of bacterial targets. Regardless of the fact that we model many of our discussions around bacterial systems, it should be emphasized that the lessons of this chapter are applicable to gene expression profiling experiments in all organisms.

Primary sources of experimental and biological variation

Differences among samples

It is, of course, desirable to minimize extraneous biological and experimental variables as much as possible when analyzing gene expression profiles obtained under two defined experimental conditions such as a temporal or treatment gradient, or between two different cell sample types or genotypes [1, 2, 3, 4]. In Chapter 7, we compare the gene expression profiles between two genotypes, lrp+ and a lrp− strains of E. coli.