Isolation of total RNA from roots of soil-grown plant

AVICO, E.; ACEVEDO, M.; RUIZ, O.; SANSBERRO, P.

Congreso; International Congress of Plant Molecular Biology, ICPB 2015; 2015

The extraction of pure nucleic acids from root tissues is generally difficult mainly because of its high fiber content which affects the process of cell lysis. Likewise the high concentration of storage compounds, like carbohydrates and secondary metabolites, also negatively affects the process of purification. Likewise, in cases in which the extraction must be made from roots that grow in the soil, the presence of enzymatic inhibitors such as fulvic and humic acids negatively affect the quality of it. The aim of this study was to evaluate the suitability of total RNA extracted from roots of Ilex paraguariensis, a non-model woody plant species, by probing commercial kits. Roots were collected from three potted plants grown under controlled environmental conditions. Extractions were performed by triplicate using SpectrumTM Plant Total RNA kit (Sigma®), RNeasy® Plant Mini Kit (QIAGEN®), TRI Reagent® (Molecular Research, Inc.) and SV Total RNA Isolation System (Promega®). Integrity was checked by agarose gels electrophoresis. Purity was performed by spectrophotometric measurements using A(260-320)/(280-320) and A(230-320)/(260-320) ratios. Samples with A(260-320)/(280-320) ratio upper than 1.8 were quantified at 260nm. cDNA obtained by retrotranscription was used to amplify a β-tubulin sequence by qPCR. The quality ratio of RNA obtained with TRI Reagent® was not enough to quantify. SV Total Isolation Kit had the lowest yield with acceptable quality. RNeasy® Plant Mini Kit and Plant Total RNA Kit SpectrumTM provided higher quality and quantity of RNA; however, the latter disclosed the best performance. All displayed good integrity. qPCR was positive for all samples tested. Considering the quality, purity and integrity of the resulting extract, Total RNA kit (SigmaTM) is considered the best suited method to extract total RNA from Ilex paraguariensis roots of soil-grown plants.