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

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 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.

Figure 1

Detection of RNA extracted with different methods, Poly(A)⁺ RNA and the cDNA quality using agarose gel electrophoresis. Total RNA from jatropha immature seeds: (A) RNA extracted with method I(Lane i), RNA extracted with method II (Lane ii) and RNA extracted with Qiagen RNA Mini Kit (Lane iii). (B) Poly(A)⁺ RNA purified from the total RNA using modified method II. (C) Double strand cDNA (ds cDNA) after size fractionation with column chromatography. The RNA and 1-kb DNA marker were from New England Biolabs. The image is representative of four independent experiments.

Figure 2

Evaluation of jatropha seed cDNA library. Poly(A)⁺ RNA was purified from total RNA extracted with method II and used for cDNA library construction using CloneMiner™ cDNA Library Construction Kit (Invitrogen). Plasmid DNA of 20 positive clones was digested with BsrG1 enzyme (New England Biolabs) and electrophoresed on 1% agarose gel to determine average insert size of cDNA. (Lane 1) 1-kb DNA marker (New England Biolabs); (lane 2) vector pDONR™ 222 (Invitrogen); (Lanes 3-22) randomly picked cDNA clones. Band at size 2.5 Kb is the vector backbone. cDNA insert of the clone in lane 3 has similar size as that of vector backbone, which did not separate in this gel electrophoresis.

Figure 3

Northern hybridization of jatropha immature seed RNA with Ketoacyl ACP reductase (KAR) probe. RNA extracted with method I and II from jatropha leaves and seeds was fractionated on 1.2% agarose-formaldehyde denaturing gel and blotted onto Hybond-N+ nylon membrane. Stained blot was photographed and then hybridized with Ketoacyl ACP reductase (KAR) probe labelled with [32P]-dCTP. The hybridized bands were detected by exposing filters to KODAK Biomax MS Autoradiography Film at -80°C. The image is representative of four independent experiments.