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David Weaver et al.
two samples, we [1–3] and others [2–6] used these technologies to assess the mRNA expression status of candidate genes within the genomic segments prioritized by mapping studies for hypertension and metabolism-related phenotypes [7]. Some of these mapping studies led to the detection of differentially expressed genes as potentially novel candidate genes for blood pressure regulation in rats. A good example is the prioritization of the gene coding for the nuclear receptor 2, factor 2 [1]. This gene located on rat chromosome 1 was prioritized through a rat microarray experiment [1] and many years later also prioritized in human hypertension through a reanalysis of a genome-wide association study [8]. During the decade since the microarray platform came into existence, this technology has not only expanded in terms of its ability to detect and analyze transcriptomes comprising of mRNAs, but has grown dynamically to encompass the analysis of noncoding RNAs such as microRNAs and long noncoding RNAs (LncRNAs), and PiwiRNAs. Given that very little is known regarding the role of these new classes of noncoding RNAs in the genetics of hypertension and that the basic principles and methodologies associated with a microarray experiment for either mRNAs or noncoding RNAs remains essentially unchanged, the microarray technology can be predicted to be a mainstay in the quest for genetic elements controlling blood pressure. Therefore, in this chapter, we chose to describe the methods to conduct and analyze a microarray experiment. The chapter also catalogs information on pertinent websites that we have accessed during our studies for analyzing our datasets.
2 Sample Preparation for Microarray 2.1 Total RNA Isolation
The quality of the RNA is essential to the overall success of the analysis. Since the most appropriate protocol for the isolation of RNA can be source dependent, we recommend using one of the commercially available kits designed for RNA isolation such as TRIZOL (Life technologies) or QIAzol (QIAGEN). RNA thus obtained is of poor quality for hybridization experiments. A cleanup procedure using an RNA cleanup kit such as RNeasy Kit (Ambion) is important.
2.2 Reagents and Materials Required
1. TRIZOL Reagent: Invitrogen Life Technologies, P/N 15596- 018, or QIAzol™ Lysis Reagent: QIAGEN, P/N 79306. 2. RNeasy Mini Kit: QIAGEN, P/N 74104. 3. 10× TBE: Cambrex, P/N 50843. 4. Absolute ethanol (stored at –20 °C for RNA precipitation; store ethanol at room temperature for use with the GeneChip Sample Cleanup Module and IVT cRNA Kit). 5. 80 % ethanol (in DEPC-treated water) (stored at −20 °C for RNA precipitation; store ethanol at room temperature for use with the GeneChip Sample Cleanup Module).
