Rapid Cycle Real-Time PCR - Methods and Applications [antikvár]

Housekeeping Genes: A Gold Standard? Onno Barker*, Daphne C. Timmer Introduction Gene expression studies that aim at precision require normalization to an internal control or "housekeeping" gene. The greatest challenge in choosing an appropriate housekeeping gene is maintaining expression consistency during treatment. Many genes can, in principle, be used as housekeeping genes [1]; however, the two most commonly used are glyceraldehyde-3-phophate dehydrogenase (GAPDH) and (3-actin. Unfortunately, these genes are not as constant in their expression as one would think (or hope) and this particular problem has resulted in many recent publications [2-10]. (3-actin shows a diurnal rhythm and the expression level changes when treating with certain hormones [4]. In addition, the presence of pseudogenes can be confounding when there is, even a little, DNA contamination in the RNA preparation [11]. Similar difficulties can occur with the well-known housekeeping genes GAPDH, elongation factor 1 alpha (EFla), and cyclophilin [11]. Usually, an adequate housekeeping gene can be found in a controlled system (e.g., cell culture) using a commercial kit or the "trial and error" method. However, more challenges arise when working with samples from patients who undergo a variety of treatments [12], because it is impossible to manipulate conditions or repeat the experiment. Similar difficulties arise when the expression of a specific gene is studied in animals throughout the day. Therefore, this chapter focuses on two specific experimental designs: (1) the expression of four different housekeeping genes studied in patient tissue samples, and (2) the expression of three housekeeping genes studied during a 12-h light and a 12-h dark cycle in rat liver.