Abstract
A new and simple boiling method for extracting chrysanthemum stunt viroid (CSVd) RnA is described. Fresh leaves of CSVd-infected chrysanthemum were homogenized in an equal volume of CTAB buffer then centrifuged. The supernatant was boiled for three min then centrifuged again. Transparent supernatant of boiled sample was collected. CSVd-RNA was then purified by the following procedures: the supernatant (300 μl) was added to 1/10 volume of 3M sodium acetate and an equal volume of isopropyl alcohol, transferred gently to a microcentrifuge tube containing 400 μl 2M LiCl, incubated at room temperature for 10 min and centrifuged. Northern blot hybridization analysis showed that the extracted CSVd-RNA survived although it was partially broken by boiling. This was confirmed by RT-PCR amplifying about 350 base pairs cDNA, showing the full length of CSVd. Dot blot hybridization analysis indicated that the boiling extraction method has a higher detection sensitivity than the guanidine thiocyanate/phenol extraction method. It is also concluded that boiling is a safe method that does not require toxic reagents such as phenol and chloroform.
