An improved method for total RNA isolation from recalcitrant loquat (Eriobotrya japonica Lindl.) buds

Loquat buds during floral differentiation which contain large amounts of polysaccharides,proteins and secondary metabolites were not amenable to conventional rna isolation protocols. here a concise and efficient rna isolation protocol based on cetyl trimethyl ammonium bromide (ctab) and lithium chloride (licl) named improved ctab-licl protocol was developed. the rna isolated by this improved protocol was not only of high purity and integrity (a260/280 ratio was range from 1.85 to 2.0 and a 260/230 ratio was over 2.0),but also high yield as 400-600 μg of total rna per gramme fresh tissue,which was 6 to 10 folds more than that by the improved ctab i or ii protocols assessed. these results depended on the following crucial improved steps: the addition of 2 ml (3m) potassium acetate to further deposit polysaccharides,three times efficient separation of total rna from polysaccharide and dna residues with 2 m lithium chloride (licl) which was never applied in the traditional ctab-based protocols,the reduction of phenol-chloroformisoamylalcohol extraction times (only once) to avoid the great loss of total rna. further,the quality of total rna isolated was verified to be competent for the reverse transcription-polymerase chain reaction (rt-pcr) and northern blot analysis with ejtfl1-1 and ejtfl1-2 genes related to floral bud differentiation. moreover,this improved protocol is cost-saving and reduces the risk of chemical carcinogen to operator as the abandon of diethyl pyrocarbonate (depc).