An improved method for total rna isolation from recalcitrant loquat (Eriobotrya japonica lindl.) Buds | [Pakistan Journal of Botany • 2011]

Author(s):

1. Huwei Song: College of Horticulture, South China Agricultural University, Guangzhou 510642, P.R. China Jiangsu Key Laboratory of Eco-Agricultural Biotechnology around Hongze Lake, Huaiyin Normal University, Huaian 223300, Jiangsu, P.R. China

2. Yuexue Liu: College of Horticulture, South China Agricultural University, Guangzhou 510642, P.R. China College of Horticulture, Shenyang Agricultural University, Shenyang 110161, P.R. China

3. Guibing Hu: College of Horticulture, South China Agricultural University, Guangzhou 510642, P.R. China

4. Yonghua Qin: College of Horticulture, South China Agricultural University, Guangzhou 510642, P.R. China

5. Shunquan Lin: College of Horticulture, South China Agricultural University, Guangzhou 510642, P.R. China

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-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).

Page(s): 1163-1171

DOI: DOI not available

Published: Journal: Pakistan Journal of Botany, Volume: 43, Issue: 2, Year: 2011

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