6 protocols
nucleic acid hybridization to array protocol
Overall 25,262 transcripts were represented on the microarray by 1, 2 or 3 individual probes. Hybridizations for all experimental conditions were performed in 4 replicates. Total RNA samples derived from the treatments were hybridized against the pooled control respective to their origin. The microarray hybridization procedure was carried out with 300ng of cyanine-3 and cyanine-5 labelled cRNA for 17h at 65°C. Control/control hybridization were performed, each component of the pooled control (LP 2°C, LP 7°C, LP 12°C) was hybridized against the pooled control to mitigate dye bias effects. Subsequently microarray disassembly and wash procedure followed as described by the manufacturer’s instructions (Agilent).
nucleic acid extraction protocol
Frozen sporophytes were ground in liquid nitrogen and transferred to 2.0 ml Eppendorf tubes. 2 ml extraction buffer (2% CTAB, 1 M NaCl, 100 mM Tris pH 8, 50 mM EDTA, pH 8) and 20 ?l DTT 2M were added and mixed well. The mixture was incubated at 45 °C for 10 min. One volume of chloroform: isoamylalcohol (24:1) was added and mixed vigorously for 10 min. The tubes were centrifuged for 20 min at 20 C and 12 000 rcf. 750 ?l of the aqueous phase were transferred into a new tube. 0.3 volumes of EtoH 100% were added and mixed gently by inverting the tube. One volume of chloroform: isoamylalcohol (24:1) was added and a second chloroform extraction followed. 500 ?l of the supernatant were transferred to a new cup and total RNA was extracted using a Quiagen Plant Mini Kit (Qiagen, Hildesheim; Germany) according to manufactures protocol for RNA Extraction including on-column DNA-digestion. Quantity and purity of the extracted RNA was determined by a NanoDrop ND-100 spectrometer (PeqLab, Erlangen, Germany). For verifying the integrity of the RNA the RNA Nano Chip Assay with the 2100 Bioanalyzer device (Agilent Technologies, Böblingen, Germany) was performed.
growth protocol
Saccharina latissima (Lane) sporophytes were raised from gametophyte cultures, which were established from spores of fertile sporophytes collected by SCUBA diving in Kongsfjorden, (79°N; 11°E; Svalbard, Norway; AWI culture numbers: 3123, 3124). Male and female gametophytes were fragmented together, transferred to petri dishes filled with Provasoli enriched seawater (Starr & Zeikus, 1993) and cultured at 10 ± 1°C and 30 µmol photons m2*s-1 photosynthetically active radiation (PAR) at 18h light : 6h dark period . After 2 weeks developing sporophytes were transferred to aerated 5l culture bottles and grown with twice weekly changes of enriched seawater until they reached a size of 5-7cm.
nucleic acid labeling protocol
Total RNA was labelled with the Agilent two-colour Low Input Quick Amp Labeling kit (Agilent Technologies, Waldbronn, Germany). For the pooled control sample 700 ng RNA from every low PAR treatment was mixed together prior to the labeling in equal molar concentrations. RNA from stress treatments was labeled by fluorescent complementary RNA (cRNA) synthesis with cyanine-5-CTP, control sample RNA was labeled with cyanine-3-CTP. Agilent RNA Spike-In Mix (Agilent) was added to 200 ng of total RNA prior to the labelling. On account of the extensive length of 3`untranslated regions (UTRs) occurring in brown algae, cDNA synthesis was performed using a blend of T7 nonamer primer and T7 promoter primer in equal molarity. cRNA synthesis and purification of labelled RNA was conducted following the two-colour Low Input Quick Amp Labeling kit protocol (Agilent).
treatment protocol
Young sporophytes were exposed for 24 hours in environmentally controlled rooms (2, 7 and 12 C ± 1 C) to high photosynthetically active radiation (PAR) alone and low PAR in combination with UV-radiation (UVR). Experimental light conditions are shown in table 1. All experiments were conducted with 5 replicates. To distinguish the effects of different wavelengths ranges, the experimental units were covered with cut-off filter foils permeable to wavelengths: (1) 400- 700 nm (URUV Ultraphan UV farblos, Difrega, Germany) for photosynthetically active radiation (PAR) treatments, and (2) 295-700nm (URT 140 Ultraphan UV farblos, Difrega) for PAR + UVA + UVB treatments. PAR was provided by 3/5 Osram daylight fluorescent tubes (Biolux, 36W; Osram, Germany) and measured by using a LI- 250 light meter (LI-COR, Lincoln; USA). UV irradiance was generated by 3 fluorescent tubes (UV A-340, 40W; Q-Panel, USA) and determined with a Solar Light PMA-2100 (Solar Light; PA, USA).