An improved method for RNA isolation and cDNA library construction from immature seeds of Jatropha curcas L

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.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 applications. This method takes less time for RNA extraction and is equally effective for other tissues where the quality and quantity of RNA is highly interfered by the presence of fatty acids, polysaccharides and polyphenols.Efficient isolation of high quality and quantity of total RNA from plants is highly desirable for the construction of a good-quality cDNA library. Several commercial reagents and kits are available for isolating RNA from plants (e.g., Trizol, Gibco-BRL Life Technologies; RNeasy plant kit, QIAGEN), but they are not always effective for all plant tissues, particularly for developing seeds of oil rich plants. Seeds from oilseed plants contain high levels of extractable lipids, polysaccharides and phenols and many other secondary metabolites that could interfere with RNA extraction, by degrading or co-precipitating with the extracted RNA [1-3]. Thus most RNA isolation methods either result in very low yields of RNA or form complexes with these contaminants resulting in low quality poly(A)+ RNA unsuitable for first strand cDNA synthesis and RT-PCR [4]. Such undesirable outcomes prompted researchers to develop new improved protocols for RNA extraction from