Stability investigation of air-dried olive ribo nucleic acids for metavirome studies

The application of ribo nucleic acids for molecular studies requires high integrity and quality of extracted total RNA samples. In addition, the need to transfer RNA samples at room temperature without special treatments such as ice and liquid nitrogen storage according to international transport laws highlights the importance of low cost alternative methods such as RNA air-drying, lyophilisation and transportable agents. In this study, the quality and quantity of air-dried RNA samples from leaf, petiole and bark tissues of different olive genotypes using several RNA extraction methods were compared with lyophilized ground leaves and RNAlater-stored tissue samples before precipitation. The quality of RNA and prepared libraries were checked by several techniques including agarose and polyacrylamide gel electrophoresis, Agilent quality control, RT-PCR amplification of housekeeping and viral genes and high throughput sequencing.

Although RNA value varied amongst cultivars, RNA extraction with TRIzol™ Reagent in fresh extractions and samples stored in RNAlater before RNA extraction resulted in 455.26 ng/µL and 63.46 ng/µL (mean value of cultivars) as the highest RNA concentration averages, respectively. RNA samples extracted by TRIzol™ Reagents and stored for a short term at - 80 °C before air-drying showed the third highest concentration (44.87 ng/µL). The synthesized cDNAs quality for PCR amplification of housekeeping genes (Rbc 1 and Nad 5) and partial genomes of Arabis mosaic virus and Cucumber mosaic virus showed satisfactory results in RNA samples extracted by TRIzol™ Reagents despite its variation amongst cultivars.

Considering the difficulties in the extraction of high quality and quantity RNA in olive for molecular analyses, this study demonstrated that RNA extraction method based on TRIzol™ Reagent can be considered for virobiome studies of both fresh and air-dried samples.