RNA III

After total RNA isolation and a successful DNA digestion, we need to purify mRNA from our total RNA extract. Total RNA consist of somewhere between 1-5% mRNA (organism dependent) and the other ~95% contains ribosomal RNAs, TRNAs and other small RNAs. Eukaryotic systems have an advantage over prokaryotic systems due to a specific biochemical advantage at the 3’ end of each mRNA transcript. This advantage is a polyadenylation event, or a 15-30 base pair adenine rich tail (consistent adenine repeat) at the end of each transcript. This is advantageous because we can design a “handle” that will, without fail, hybridize to our polyadenylated transcript. In order to isolate an efficient amount of mRNA for RNA-seq (~500ng) you need to start with a large amount of total RNA (remember that only 5% of total RNA are mRNAs), something around 3mg of total should be efficient. Quality of mRNA input into an RNA-seq experiment is extremely important. If care is not taken in the cleanup step, you might as well start over.