RNA I

The overarching goal of my Transcriptomics (RNA sequencing) class is to provide an outline, or protocol to send samples for RNA-seq that would work for anyone working in our system. This is a great idea because we are getting a wide range of protocols covering a wide range of systems. All eukaryotic systems are virtually identical, however, the prokaryotic crowd encompasses aerobic and anaerobic organisms with radically variable needs.

The starting place for an RNA-seq protocol begins with total RNA isolation. This process differs between plants and animals due to the presence of a cell wall in plants. Plant RNA isolation is a fairly simple process, if using a kit. Before one can begin to isolate this fragile nucleic acid, certain procedures must be taken and potential problems must be addressed. First, RNA is single stranded, thus lacking the hydrogen-bonding matrix that DNA, a double stranded nucleic acid contains. This property makes RNA very fragile. Second, our skin, breath, and hair are riddled with proteins called RNASes. These are proteins that target RNA and chop the transcripts into untranscribable messages, thus destroying the polynucleotide. While isolating RNA, one must not take these two considerations lightly. We ALWAYS use gloves and RNAse/DNAse free materials including water, tubes, isolation columns, and all solutions used throughout the isolation process, as well as any downstream single stranded nucleic acid application.