Protocol adjustment improves the extraction of high-quality total RNA from common bean stems infected by Sclerotinia sclerotiorum

Abstract

The Straw Test is an assay developed to evaluate the resistance of common bean to white mold, in which the plant stems are inoculated and the symptoms of the disease are monitored. It is plausible to admit that investigating gene expression in pathogen-infected tissues may be strategically interesting. However, obtaining a quality RNA is a basic requirement for this purpose. Therefore, the objective of this study was to evaluate adjustments in protocols of commercial kits in the expectation of improving the quality of RNA obtained from bean stems. For this, plants of two lines were inoculated and the stems pathogen-infected were collected 72 hours after. For RNA extraction, two commercial reagents were used following the manufacturer’s recommendations and then following adaptations in these protocols. In particular, the proposed modifications relate to volumes of supernatant recovered in purification steps, additional step of chloroform purification and extended time for nucleic acids precipitation. The obtained RNA was analyzed by spectrophotometer, electrophoresis and bioanalyzer, then converted into cDNA and subsequently submitted to PCR. From the obtained data, it was observed that the adaptations made in the protocols contributed to better results and that, when the indicative values of RNA quality are guaranteed, the subsequent reactions are more pure, precise and representative.