A protocol to remove colored metabolites and other inhibitors from plant tissues to facilitate RNA isolation suitable for downstream applications

Abstract

This work developed a protocol to remove colored metabolites and other interfering substances to facilitate RNA isolation. These metabolites otherwise hinder RNA isolation and downstream applications. The developed protocol used sodium dodecyl sulphate, ethylenediamine tetra-acetic acid, and ethanol in a definite ratio that removed the said metabolites from the tissue and aided isolation of RNA using the existing methods. The protocol was developed for red colored roots of Arnebia euchroma and was extended to other colored tissues [rhizome of Rheum australe (Himalayan Rhubarb) and taproot of Daucus carota (purple carrot)] with success. Without inclusion of our protocol, the existing methods could not isolate good quality RNA from these tissues. RNA isolated by the developed protocol had A(260/280) ratio of 1.88-1.93, A(260/230) ratio of 1.94-2.0, and RNA integrity number of 6.3-8.0. RNA was amenable to downstream applications such as reverse-transcription polymerase chain reaction and primer extension assay.