an improved method for rna isolation and cdna library construction from immature seeds of jatropha curcas l一种改进方法,rna隔离和不成熟的互补脱氧核糖核酸图书馆建设l麻风树的种子.pdfVIP

Sangha et al. BMC Research Notes 2010, 3:126 /1756-0500/3/126 S HO RT REPORT Open Access Short Report An improved method for RNA isolation and cDNA library construction from immature seeds of Jatropha curcas L 1,2 1 3 1 Jatinder Singh Sangha* , Keyu Gu , Jatinder Kaur and Zhongchao Yin Abstract Background: RNA quality and quantity is sometimes unsuitable for cDNA library construction, from plant seeds rich in oil, polysaccharides and other secondary metabolites. Seeds of jatropha (Jatropha curcas L.) are rich in fatty acids/lipids, storage proteins, polysaccharides, and a number of other secondary metabolites that could either bind and/or co- precipitate with RNA, making it unsuitable for downstream applications. Existing RNA isolation methods and commercial kits often fail to deliver high-quality total RNA from immature jatropha seeds for poly(A)+ RNA purification and cDNA synthesis. Findings: A protocol has been developed for isolating good quality total RNA from immature jatropha seeds, whereby a combination of the CTAB based RNA extraction method and a silica column of a commercial plant RNA extraction kit is used. The extraction time was reduced from two days to about 3 hours and the RNA was suitable for poly(A)+ RNA purification, cDNA synthesis, cDNA library construction, RT-PCR, and Northern hybridization. Based on sequence information from selected clones and amplified PCR product, the cDNA library seems to be a good source of full- length jatropha genes. The method was equally effective for isolating RNA from mustard and rice seeds. Conclusions: This is a simple CTAB + silica column method to extract high quality RNA from oil rich immature jatropha seeds that is suitable for several downstream applicat