Isolation of High Quality RNA for High Throughput Applications From Secondary Metabolite Rich Crocus Sativus L

Abstract

Isolating high quality RNA is a basic requirement while performing high throughput sequencing, microarray and various other molecular investigations. However, it has been quite challenging to isolate RNA with absolute purity from plants like Crocus sativus that are rich in secondary metabolites, polysaccharides and other interfering compounds which often irreversibly co-precipitate with the RNA. While many methods have been proposed for RNA extraction that include CTAB, TriZol, SDS based methods, they invariably yield less and poor quality RNA. In the present study we made certain changes in the available protocols including modifications in the extraction buffer and procedure viz-a-viz solutions used for precipitation of RNA. Our method led to the isolation of clear and non-dispersive total RNA with an RNA Integrity Number (RIN) greater than 7.5. The quality of the RNA was further assessed by qPCR based amplification of mature miRNAs such as Cs-MIR166c and Cs- MIR396a. In conclusion, the study describes an efficient method of RNA extraction that is highly ideal for high throughput sequencing of small RNAs.