ISOLATION OF TOTAL RNA FROM ROOTS OF POTTED ILEX PARAGUARIENSIS: ASSESSMENT OF FOUR METHODS.

AVICO E.; ACEVEDO R. M.; RUIZ O.A.; SANSBERRO P.

ROSARIO

Congreso; L REUNION ANUAL SAIB; 2014

SOCIEDAD ARGENTINA DE INVESTIGACION EN BIOQUIMICA Y BIOLOGIA MOLECULAR

There are no published studies that indicate appropriate methods for isolation and purification of RNA from roots of yerba mate. The aim of this study was to evaluate the suitability of total RNA extracted from roots of yerba mate grown in soil by 4 commercial methods for downstream molecular biology experiments. Roots were collected from 3 potted plants. Extractions were performed by triplicate with: SpectrumTM Plant Total RNA kit (Sigma®), RNeasy® Plant Mini Kit (QIAGEN®), TRI Reagent® (Molecular Research, Inc.) and SV Total RNA Isolation System (Promega®). After digestion with DNase I, integrity was checked by agarose gels electrophoresis. Quality was assessed by (260-320)/(280-320) and (230-320)/(260-320) ratios. Samples with (260-320)/(280-320) ratio greater than 1.8 were quantified spectrophotometrically. cDNA obtained by RT 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, the second one showed best performance. All them showed good integrity. qPCR was positive for all samples tested. Considering quality, integrity, performance and cost, the commercialized kit by Sigma® is the best method to extract total RNA from studied tissue.