Sample labeling: An overview

It is not easy to write a critical review of the methods available for labeling RNA and DNA "extracts" for microarray studies. There are a number of reasons for this: Suppliers of the reagents and kits used for this purpose do research and development, quality control, and validation and then they provide a hard-wired, "optimized" product. They often give few details about the compositions of these products, are inclined to put the best face they can on what they sell and gloss over any deficiencies, and have no interest in paying for direct comparisons of their product to those of other companies. These comparisons can be expensive to perform, and there are few good examples in the literature. When comparative experiments have been done, it is not clear that each of the individual methods tested was executed with equal proficiency. Many experiments can be required to determine how best to hybridize any given labeled extract to a particular array and how to block, wash, and postprocess (e.g., stain) the array so that the signal-to-noise ratio is maximized. In addition, authors of comparative studies used different arrays, technical protocols (some of which are out of date), experimental designs, and analyses. Finally, some new techniques, which seem quite promising, have been employed so little that their strengths and shortcomings are difficult to assess. Given these constraints, this chapter attempts to (1) outline the steps involved in labeling extracts, (2) touch on problems that remain to be solved, and (3) describe and evaluate a variety of labeling methods. Methods used for studying gene expression as opposed to genomic DNA are predominant in this chapter, but the latter are mentioned in passing. Nucleic acids have to be extracted from cells or tissues before they can be labeled. Next, if there is too little template to label efficiently, the RNA or DNA must be amplified. Finally, the unamplified or unamplilied products have to be tagged so that they can be detected. This last step can occur before the products are hybridized to elements on the array or afterwards, and techniques are available that allow the user to perform signal amplification as well as template amplification.