A comparison of six total RNA isolation methods for diagnosis of GYSVd-1 (Grapevine yellow speckle viroid-1) on vitis vinifera L. leaves

Isolation of high quality RNA from plant tissues is one of the most critical steps for the successful application of diagnostic tests such as reverse transcription polymerase chain reaction (RT-PCR), northern blotting, microarray hybridization. The presence of inhibitors such as secondary metabolites, phenolic compounds and RNAses can cause inaccurate and undesirable results. Grapevine is rich in a wide range of metabolites which interfere with RNA isolation. From this point of view, we researched six different total RNA extraction methods on leaves of Vitisvinifera L. to find the best one that contribute the purity and high quality. The methods tested are silica-capture, modified silica-capture, commercial kit, the new combined, lithium chloride and citric buffer. RNA quality was analyzedspectrophotometrically by nanodrop, agarose gel electrophoresis and RT-PCR. As a result of all, it is clear that the most suitable TNA isolation protocol is the new combined method which experienced and named firstly by us, in terms of RNA purity, concentration, less time consuming of isolation step and achievement on detection of GYSVd-1. © 2014, Pakistan Botanical Society. All rights reserved