Natarajan et al. BMC Genomics 2010, 11:606 http://www.biomedcentral.com/1471-2164/11/606 Page 8 of 14 Figure 4 ORF analysis. The unigenes that did not show significant similarity to any genes in the protein database at NCBI were taken for ORF prediction using ORF finder tool at NCBI to identify potential full-length unigenes. Putative ORFs were predicted for 32 unigenes and 14 of them were found to be potential full-length unigenes. J. curcas for the first time. From these sequences, 7009 unigenes were identified which included 6233 potential full-length genes. These genes encoded for diverse biolo- gical functions in J. curcas including oil biosynthesis, stress response, flavanol biosynthesis etc. These genes will serve as invaluable resource for the genetic engi- neering to modify oil composition and to increase oil content, seed yield, pest and disease resistance to make jatropha more suitable for biodiesel production and profitable to farmers. Gene discovery from other tissues of J. curcas are being attempted by using next genera- tion sequencing technology. Methods Collection of Seeds Seeds in different developmental stages were collected from Jatropha curcas fruits which were between 0.5 and 2.5 cm in diameter and green in colour (Figure 1). The seeds were flash frozen in liquid nitrogen and stored at -86°C until further use. Isolation of Total RNA Total RNA from developing seeds was prepared using Trizol reagent following the manufacturer’s protocol (Invitrogen, USA). One gram of the developing seeds were ground with liquid nitrogen to a fine powder and suspended in 10 ml Trizol reagent. The suspension was mixed well, and incubated on ice for 10 min. Subse- quently, 2 ml chloroform was added to the suspension and incubated on ice for 10 min. The suspension was centrifuged at 10,000 × g for 15 min at 4°C. The aqu- eous phase was transferred to a fresh tube, and equal volume of isopropanol was added. It was incubated at -20°C for 1 h, and total RNA was recovered by centrifu- gation at 10,000 × g for 20 min at 4°C. The supernatant was discarded and the pellet was washed with 70% etha- nol, dried and dissolved in 200 μl RNase free water. The total RNA was further purified by using RNeasy Mini Kit following the manufacturer’s protocol (Qiagen, Hil- den, Germany). Quality of total RNA was checked by agarose gel electrophoresis as well as OD260/OD280 ratio before using it for cDNA synthesis. cDNA Synthesis and Normalization Synthesis of full length cDNAs from total RNA and nor- malization were performed as described by Patanjali et al. [59] and Soares et al. [60] with slight modifications. Two cDNA preparations were made using the same total RNA. One was used as tester cDNA and the other was used as driver cDNA. First strand tester cDNA and driver cDNA were synthesized using tester3 primer (CAGTGGTAT- CAACGCAGAGTGGCCGAGGCGGCCT15) and driver3 primer (GGGATAACAGGGTAATGGCCGAGGCGGC CGACATGT15), respectively. Tester adaptor (GTAAC- TAGGCCGTAATGGCCACTCTGCGTTGATACCAC TG) and driver adaptor (GGCCGTAATGGCCTCGC- TACCTTAG GA) were ligated to the 3’end of the newly synthesised first strand tester cDNA and driver cDNA, respectively. These adaptors will ligate only to the 3’end because, they were 5’phosphorylated and 3’blocked. Dou- ble strand tester cDNA was prepared by low cycling PCR using tester3 primer and phosphorylated tester5 primer (CAGTGGTATCAACGCAGAGTGGCCATTACGGCC- TAGTTACGGG) which is complementary to the 5’tester