Efficient Method for Isolation of High-Quality RNA from Psidium Guajava L. Tissues

Abstract

Background: Acquiring high-quality RNA in sufficient amounts is necessary in plant molecular biology and genetic studies. Several methods of RNA extraction from plants are available in the literature, mainly due to the great biochemical diversity present in each species and tissue, which can complicate or prevent the extraction. Psidium guajava (Myrtaceae family) is a perennial fruit tree of medicinal and economic value; nevertheless, only few molecular and omics studies are available for the species. One reason for this fact is the difficulty in obtaining the RNA due to the content of the samples, which are rich in polyphenols, polysaccharides and secondary metabolites. Furthermore, there is still no tested or standardized method available for the isolation of RNA from guava or Psidium samples, which hampers advances in the genus.Results: Here we compare the quality and yields of RNA isolated using six extraction protocols: two based on the application of cetyltrimethylammonium bromide (CTAB) lysis buffer, two commercial kits (PureLink™ RNA Mini Kit and RNeasy® Plant Mini Kit), one using the TRIzol® reagent, and one applying guanidine thiocyanate lysis buffer followed by organic phase extraction. RNA integrity, quality and yields were assessed by agarose gel electrophoresis and spectrophotometry. The CTAB-based method provided the highest RNA yields and quality for five different tissues (flower bud, immature leaf, young leaf, mature leaf and root), genotypes and stress conditions. For the most efficient protocol, the average yield of RNA from guava leaf was 210.4 μg/g of tissue, and the A260/A280 and A260/A230 ratios were 2.1 and 2.2, respectively. RT-PCR analysis demonstrated that the purity of the samples was sufficient for molecular biology experiments.Conclusion: CTAB-based methods for RNA isolation were found to be the most efficient, providing the highest RNA yields and quality for tissues from P. guajava. Additionally, they demonstrated to be compatible for downstream RNA-based applications, besides showing the advantages of lower cost and time investments.