Northern blot analysis to investigate the abundance of microorganisms

Modern molecular microbial ecology has its origins in the analysis of informative macromolecules. Zuckerkandl and Pauling proposed that certain macromolecules are relatively free from evolutionary pressure and may be considered a molecular document of the evolutionary history of the organism that carries the molecule. In their paper, they proposed that the sequence difference of a molecule is proportional to the evolutionary distance between the organisms; the greater the sequence differences the greater the evolutionary distance. A significant breakthrough with this approach in microbial systematics resulted from the work of Woese and Fox who used oligonucleotide cataloguing of 16S-rRNA to delineate the phylogenetic relationships between microorganisms. By using this approach, it was possible to demonstrate that all life on earth could be divided into three kingdoms: eukarya, procarya and archaea. The unique findings of this research was that the archaea, made up of many methanogenic and thermophilic microorganisms, were probably the most ancient life forms on earth and were not bacteria at all. One of the first applications of rRNA genes was the recovery of unique 5S-rRNA sequences from the Yellowstone hot spring. Even though the statistical utility of the short 5S sequences was limited, it demonstrated that there was a great deal of uncultured diversity within the ecosystem. This uncultured diversity was demonstrated to be highly significant when clone libraries were constructed from the Yellowstone hot spring. Universal PCR primers were used to amplify 16S-rDNA from the microbial community, and these mixed amplicons were cloned into a vector. Each insert, potentially representing a different species, was sequenced giving a snapshot of microbial diversity in the sample. A unique feature of the rRNAs is that they are hierarchical molecules. This means that there are regions where the molecules is highly conserved, others where the sequence is variable, and even areas known as hyper-variable regions which have a high degree of sequence variation. As a result of this structure, it is possible to design signature oligonucleotide probes varying in length from about 15 to 30 nucleotides that are diagnostic of microorganisms at the kingdom, domain, genus and even species level. These signature sequences can be used in a variety of applications such as PCR analysis, construction of clone libraries or direct probing of bulk rRNA. In this chapter, I provide detailed protocols for the analysis of extracted rRNA and give detailed procedures that must be followed to do northern blot analysis of bulk RNA extracted from the rumen