PhD thesis - Biologisk Institut - Københavns Universitet

17 Material and methodsTable 1. List of species investigated in the course of the PhD project by fluorescence markers;Serotonin and FMRFamide – neurotransmitters/peptides; Phalloidin – F-actin of the musculature; Dapi(4’, 6-diamindino-2-phenylindole) – cell nuclei marker, EdU (5-ethynyl-2’-deoxyuridine) –proliferating cells.Species (Family) Neurogenesis Myogenesis Cell nuclei,Themiste pyroides(Themistidae)Thysanocardianigra (Golfingidae)Phascolosomaagassizii(Phascolosomatidae)Serotonin,FMRFamide(chapter I)Serotonin,FMRFamide(chapter I)Serotonin,FMRFamide(chapters I-III)F-actin(chapter IV)F-actin(chapter IV)F-actin(chapter IV)cell proliferationDapi, EdU(chapter IV)Dapi, EdU(chapter IV)-RNA isolation and cDNA synthesisTotal RNA was purified from Themiste pyroides embryos at 15, 28, 40, 48, 62, 72, 84,and 111 hours post fertilizaton (hpf) (miRCURY RNA isolation kit, Exiqon, Vedbaek,Denmark). cDNA samples were synthesised by reverse transcription (RETROsript,Ambion, Woodward St. Austin, TX, USA), and stored at -20 °C until use.Degenerate PCRTo clone homeobox genes, a range of degenerate primers were designed referring toMartinez et al. (1997), Nederbragt et al. (2002), Seaver et al. (2006), and Paps et al.(2009). The sequences of the oligonucleotides used are given in Table 2.A touchdown PCR was performed with the degenerate primers given in Table2. The amplification parameters were: 3 min at 94 °C, 10 cycles of 45 sec at 94 °C, 45sec at 52 °C (every cycle -1 °C), 30 sec at 72 °C, and 30 cycles of 45 sec at 94 °C, 45sec at 42 °C, and final 30 sec extension at 72 °C. The PCR products were purified bya gel extraction kit (QIAquick, QIAGEN, Copenhagen, Denmark), and on 1 µl of thisreaction another PCR with the same parameters was performed. The samples weredisplayed by electrophoresis on a 1% agarose gel, purified, concentrated byspeedvacuum (GENEVAC EZ-2 plus , Ipswich, UK), resuspended in 10 µl distilledwater, and directly ligated into pGEM-T Easy vector using a ligation kit (Promega,