Fast and inexpensive protocols for consistent extraction of high quality DNA and RNA from challenging plant and fungal samples for high-throughput SNP genotyping and sequencing applications.

Modern genotyping techniques, such as SNP analysis and genotyping by sequencing (GBS), are hampered by poor DNA quality and purity, particularly in challenging plant species, rich in secondary metabolites. We therefore investigated the utility of a pre-wash step using a buffered sorbitol solution, prior to DNA extraction using a high salt CTAB extraction protocol, in a high throughput or miniprep setting. This pre-wash appears to remove interfering metabolites, such as polyphenols and polysaccharides, from tissue macerates. We also investigated the adaptability of the sorbitol pre-wash for RNA extraction using a lithium chloride-based protocol. The method was successfully applied to a variety of tissues, including leaf, cambium and fruit of diverse plant species including annual crops, forest and fruit trees, herbarium leaf material and lyophilized fungal mycelium. We consistently obtained good yields of high purity DNA or RNA in all species tested. The protocol has been validated for thousands of DNA samples by generating high data quality in dense SNP arrays. DNA extracted from Eucalyptus spp. leaf and cambium as well as mycelium from Trichoderma spp. was readily digested with restriction enzymes and performed consistently in AFLP assays. Scaled-up DNA extractions were also suitable for long read sequencing. Successful RNA quality control and good RNA-Seq data for Eucalyptus and cashew confirms the effectiveness of the sorbitol buffer pre-wash for high quality RNA extraction.

Determining the yield and quality of purified RNA.

After any RNA purification by any method, it is necessary to ascertain both the amount (yield) and the integrity (quality) of the RNA obtained. This is essential for any subsequent analysis and critical for any comparative analyses. This article discusses several methods for RNA quantitation and provides tips for each.

An efficient method for the extraction of high-quality fungal total RNA to study the Mycosphaerella fijiensis-Musa spp. Interaction.

Efficient RNA isolation is a prerequisite for gene expression studies and it has an increasingly important role in the study of plant-fungal pathogen interactions. However, RNA isolation is difficult in filamentous fungi. These organisms are notorious for their rigid cell walls and the presence of high levels of carbohydrates, excreted from the fungal cells during submerged growth, which interferes with the extraction procedures. Although many commercial kits are already available for RNA isolation, they do not provide, in most cases, enough amount of pure RNA to be used in upstream applications. In the present work, we propose an easy and efficient protocol for isolating total RNA from the filamentous fungus Mycosphaerella fijiensis, the most important foliar pathogen of Musa spp. varieties worldwide. In addition, we applied the proposed protocol to the isolation of total RNA from banana leaves infected with the pathogen. Our methodology was developed based on the SDS method with modifications including a carbohydrate precipitation step. The protocol resulted in high-quality total RNA, from fungal mycelium grown in PDB medium and infected banana leaves, suitable for further molecular studies. The proposed methodology is also applicable to the ascomycete fungus Passalora fulva (syn. Cladosporum fulvum).

Rayleigh light scattering study on the reaction of nucleic acids and methyl violet.

A new quantitative determination method for nucleic acids in aqueous solutions, based on the enhancement of Rayleigh light scattering of methyl violet by nucleic acids, has been developed. The sensitivity of the assay allows amounts of nucleic acids as little as 100 ng/ml to be quantitated reliably. In addition to its high sensitivity, this method has other advantages: rapidity of reaction (<5 min), simplicity of operation (one-step assay), commonality of spectrofluorimeter and reagents, stability of mixtures formed, and reproducibility. Under the experimental conditions, there is little or no interference from proteins, nucleosides, and most metal ions. Interference by a few metal ions, detergents, and some salts can be minimized by dilution. The method can also be used to determine the total amount of nucleic acids without the arduous choice of standard and difficult separation of DNA and RNA.