Comparison of RNA Purification Methods from Challenging Plant Samples and the Effect on Viroid Detection

A viroid consists of a small, single-stranded, circular RNA (240 bp - 410 bp). Viroids are known to be highly contagious, thus their rapid and reliable detection is the only way to control their spread, by eradicating all infested material at a very early stage of infection. Unlike viruses, viroids are not encapsulated in a protein coat, thus molecular hybridization techniques and PCR are most frequently used detection methods in practice. High quality plant RNA purification is a prerequisite for many downstream applications, such as reverse transcription polymerase chain reaction (RT-PCR), cDNA library construction, RNA sequencing and gene expression profiling using microarrays. Purification of high quality RNA from plant tissues is often a challenging process, due to the high concentration of polysaccharides, polyphenols and other secondary metabolites which can become complexed with RNA. In this study, a phenol/chloroform-based RNA purification method, which is known as the gold standard protocol, was compared to two market-leading spin column methods. RNA quality and quantity and small RNA recovery were all evaluated, using difficult and moderately challenging plant samples. Large differences in RNA yield and quality among protocols were found, especially with the more challenging plant samples. The effect of RNA purification method was measured by Chrysanthemum Stunt Viroid (CSVd) viroid detection. Total RNA from the silicon Carbide (SiC) column method allowed to detect low level of CSVd infection that might be beneficial to monitor an early viroid infection.