PJB-2011-127
AN IMPROVED METHOD FOR TOTAL RNA ISOLATION FROM RECALCITRANT LOQUAT (ERIOBOTRYA JAPONICA LINDL.) BUDS
HUWEI SONG1,2, YUEXUE LIU1,3,GUIBING HU1, YONGHUA QIN1 AND SHUNQUAN LIN1*
Abstract
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 A260/230 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-chloroform-isoamylalcohol 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).
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