Transcriptomics and molecular evolutionary rate analysis of the Bladderwort (Utricularia), a Carnivorous Plant with a Minimal Genome
Total RNA was extracted from U. gibba organs or whole plants exposed to the different stress conditions using TRIZOL (Invitrogen), ground with a mortar and pestle in liquid nitrogen. In the case of plants subjected to stress, 5 µg of RNA from each experimental condition was pooled to obtain a single RNA sample. First and second strand cDNA synthesis was performed with 3 ug of the total RNA mixture using Message Amp-II kit (Ambion) following the manufacturers recommendations. In the case of the three organs (traps, shoots and inflorescences), each sample was treated separately. 10-12 ng of synthesized cDNA was amplified by in vitro transcription and the resulting 70-90 ug of antisense RNA (aRNA) was purified using Qiagen RNAeasy columns (Qiagen). A second round of cDNA synthesis was performed using the mRNA as template (20 ug). cDNA synthesis was performed as described above except that random primers (mostly hexamers) were used at the first strand synthesis stage. This procedure yielded approximately 10 ug of cDNA that was purified using the DNA Clear Kit for cDNA purification (Ambion). cDNA was then treated with Ampure magnetic particles (Agencourt, Beckman Coulter) to obtain fragments of 200 – 700 pb.