General Considerations In The Analysis Of

8.1. THE IMPORTANCE OF AN RNAASE-FREE ENVIRONMENT Successful Northern blot analysis requires a preparation of RNA that is intact and relatively free from contaminants, such as protein and DNA. When working with RNA, it is also imperative to maintain an environment that is free of Rnases (ubiquitous enzymes that degrade RNA). This can be accomplished by treating deionized water and solutions that contact the RNA with 0.1% diethylpyrocarbonate (DEPC) for 24 h at 3°C prior to autoclaving (6,13). Solutions containing Tris (tris[hydroxymethyl]aminomethane) cannot be treated with DEPC. Therefore, it is recommended to reserve a bottle of Tris that is used only for RNA work and is handled appropriately. Sterile, RNAase-free tubes and pipet tips should be used at all times, and glassware should be baked or rinsed with chloroform to eliminate RNAases (6). Wash solutions are commercially available that destroy Rnases and could be useful in cleaning glassware and work stations (RNaaseAWAY, Invitrogen). The abundance of Rnases in skin necessitates the use of gloves by personnel handling RNA samples, solutions, and labware that comes in contact with samples.

8.2. TOTAL CELLULAR RNA VS MESSENGER RNA There are many variables to take into consideration when determining whether to analyze total cellular RNA or purified messenger RNA. The specificity and efficiency of the specific probe and the level of expression of the target gene contribute to the strength of the final signal observed on a Northern blot. In a preparation of total cellular RNA, only about 2.5% is actually mRNA; the majority is composed of ribosomal RNA (rRNA). The quantity of RNA that can be subjected to Northern analysis is limited to approx 30 |g by technical limitations related to the capacity of hybridization membranes for RNA binding. Therefore, detection of rare messenger RNAs (mRNAs) frequently requires utilization of purified mRNA. There are many commercially available kits for purification of mRNA, all of which take advantage of the polyadenylation signal on the 3' end of mRNAs. The basic principle involves selection of the mRNA by binding the sequential adenosine residues to a synthesized stretch of deoxythymidine residues that have been affixed to a solid support (such as cellulose or magnetic beads). The unbound ribo-somal RNA and other contaminates are washed away, and the mRNA is then eluted from the support matrix. Even rare mRNAs can be detected when as little as 1 |g of purified RNA is subjected to Northern analysis.

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