A Practical Approach, Second Edition=Ronald D. Ho.pdf

FUNCTIONAL GENOMICS AND PROTEOMICS 631on the microarray, while columns represent the samples used to hybridize to the array. The signalratios, as measures of relative changes in gene expression between experimental and controlsamples, are depicted according to a color-based scale. By convention, up-regulation of genes inthe experimental samples appears in red, whereas down-regulation is green. No change in geneexpression is represented by yellow, whereas gray or black cells indicate missing or excludeddata. 36,504. Experimental Considerationsa. Limited RNA AvailabilityTypically, running a single sample on a microarray slide requires 20 to 100 µg of total RNA. Forthose working with large tissue samples or cell cultures, these RNA requirements are usually easyto meet. However, if the biological sample of interest is derived from a very small tissue sample,such as a portion of the neural tube of a neurulation-stage mouse embryo, the amount of total RNAthat can be obtained is limited and will most likely be less than 1 µg. For those using laser-capturemicrodissection, single-sample RNA yields may be in the low nanogram range. To overcome suchproblems, it may be possible to pool a large number of individual samples so that the combinedRNA content is sufficient for labeling and hybridization. While appropriate under specific experimentalsituations, this approach is likely to mask small and subtle changes in gene expression (i.e.,have reduced sensitivity) due to a dilution effect. Most importantly, pooling of samples eliminatesthe ability to measure the biological variability that is a particularly important experimental parameterwhen working with microarrays.An alternative approach to overcome the problem is to use labeling methods that increase thespecific activity of the probes, thus compensating for the limited quantities of RNA. Examples ofsuch methods are the Micromax ® Tyramide Signal Amplification (TSA) technique (NEN LifeScience Products, Zaventem, Belgium), the HiLight Array Detection System (QIAGEN, Valencia,CA), and the 3DNA Dendrimer technology (Genisphere ® , Hatfield, PA), One must keep in mindthat none of these techniques has emerged over time as the single most powerful and reliableapproach. 23,51–53Another option to deal with limited RNA availability in gene expression experiments has beendeveloped by James Eberwine at the University of Pennsylvania. His method is based on linearamplification of mRNA by IVT from cDNA. 54,55 The cDNA used for IVT is made by reversetranscription of mRNA by use of a poly-dT oligonucleotide with a T7 RNA polymerase promotersequence. Thus, by utilizing the well-known ability of T7 RNA polymerase to bind to its promoterand synthesize RNA at a constant rate of approximately 100 bases/sec, linear amplification of RNAis achieved with high fidelity. The extent of amplification depends on the amount of template used.It is typically 40 to 200-fold with relatively large amounts of template and up to 2000-fold whenthe starting amount of template is very small, such as that obtained from single cells. 54,56This method has been successfully used for determining changes in gene expression in mouseembryos following administration of several known teratogens. In these studies, dot blots containingup to 50 genes of interest were prepared by immobilizing cDNA on nylon membranes. The probesused in these experiments were prepared by the T7-based IVT method and labeled with a radioactivenucleotide. With this approach, it has been shown that there is a significant down-regulation in theexpression of several folate pathway genes (e.g., folate binding protein-1 and methylene tetrahydrofolatereductase) in neurulation stage neural tube defect–sensitive SWV mouse embryos followingin utero exposure to valproic acid. 57 In contrast, the more resistant LM/Bc strain embryoshad an up-regulation of these same genes in response to the teratogenic valproate exposure. Theseobservations provided a plausible mechanism for relative resistance to valproate-induced neuraltube defects. Similar experimental designs were used to study additional teratogen-induced changesin expression of growth and transcription factors as well as cell cycle checkpoint genes. 58–61© 2006 by Taylor & Francis Group, LLC