Transcriptome of Lippia alba leaves using Roche 454 platform

A pool of fresh young leaves of L. alba were collected and used to RNA extraction. Total RNA was extracted according to manufactures RNeasy Midi Kit (Qiagen, Carlsbad, CA, USA) from 400 mg of fresh leaves. Total RNA was purified and analyzed on 1% agarose gel electrophoresis for quality and quantity assessment. Additionally, the samples were evaluated by Bioanalyzer 2100 (Agilent Technologies, Inc). The cDNA syntheses were carried out using the SMARTer PCR cDNA Synthesis kit and the Advantage PCR kit (Clontech, Palo Alto, CA, USA). The extension cycles of PCR were performed following manufacturer instructions. The first cDNA strand was synthesized using oligo-dT supplied by SMARTer PCR cDNA Synthesis kit using 1 g of total RNA and suspended in 40 L of TE buffer [10 mM Tris (pH 8.0), 0,1mM EDTA]. The second strand was built by LD-PCR using the Advantage PCR kit (Clontech, Palo Alto, CA, USA). The amplification program was followed by initial denaturation of 1 min at 95 C followed by 18 cycles.