array scanning protocol
Raw data were processed with the Agilent Feature Extraction Software version 184.108.40.206 (FE). Array quality was monitored using the Agilent QC Tool (v1.0) with the metric set GE2_QCMT_Feb07.
(Parameters: Scanning hardware = G2565AA DNA microarray scanner [Agilent], Scanning software = Feature Extraction Software [Agilent])
nucleic acid hybridization to array protocol
Agilent Two-Color Microarray-Based Gene Expression (Hybridization)
Agilent publication number: G4140-90050
This document describes Agilent's recommended procedures for the preparation and labeling of complex biological targets and hybridization, washing, scanning, and feature extraction of Agilent 60-mer oligonucleotide microarrays for microarray-based two-color gene expression analysis.
(Parameters: Chamber type = OTHER: Agilent SureHyb Chamber, Quantity of label target used = 825, Mass unit = Nano gram, time = 17, Tiny time unit = hours, Volume = 100, Volume unit = Micro litre, temperature = 65)
nucleic acid labeling protocol
For microarray hybridizations, RNA Spike-In Mix (Agilent, p/n 5188-5279) was added to the RNA samples prior to the labelling substances as an internal standard for hybridization performance (Agilent RNA Spike-In Kit protocol). 200 ng total RNA from
samples was reversely transcribed, and the resulting cDNA was linear amplified into labelled cRNA (two-colour Low Input Quick Amp Labeling kit, p/n 5190-2306). Incorporation of labelled cytidine 5’-triphosphate into the cRNA from the 180 and 800 uatm CO2 treatments (Cy-3) as well as for the pooled 380 uatm CO2 control
treatment (Cy-5) was verified photometrically using the NanoDrop ND1000. Labelling efficiencies were calculated as pmol dye (ng cRNA)-1.
(Parameters: Amount of nucleic acid labeled = 200, Amplification = OTHER: linear, Mass unit = Nano gram)
Cultures of Alexandrium tamarense were grown as dilute batch in 2.4 L air-tight borosilicate bottles. Filtered natural
seawater (0.2 ?m) was enriched with metals and vitamins according to the recipe of f/2-medium, except for FeCl3 (1.9 ?mol L-1), H2SeO3 (10 nmol L-1), and NiCl2 (6.3 nmol L-1). The added concentrations of NO3
- and PO43- were 100 ?mol L-1 and 6.25 ?mol L-1. Cultures were grown at a light:dark cycle of 16:8 h and an light intensity of 250 ± 25 ?mol photons m-2 s-1 provided by daylight lamps. Bottles were kept at 15°C and placed on a roller table to avoid sedimentation.
(Parameters: time unit = seconds, temperature unit = C)
Culture medium was equilibrated with air containing a pCO2 of 180 ?atm (~Last Glacial Maximum), 380 ?atm (~present-day), 800 ?atm (~2100 scenario), and 1200 ?atm (>2100 scenario).
nucleic acid extraction protocol
For RNA extraction, 500 mL of culture suspension was concentrated to 50 mL with a 10 ?m mesh-sized sieve and centrifuged at 15?C for 15 min at 4000 g. Cell pellets were immediately mixed with 1 mL 60?C TriReagent, frozen in liquid nitrogen and later on transferred to a 2 mL cryovial containing acid washed glass beads. Cells were lysed
using a BIO101 FastPrep instrument at maximum speed for 2 x 30 s, with an additional incubation of 5 min at 60?C in between. For RNA
isolation, 200 ?L chloroform was added to each vial, vortexed for 20 s and incubated for 10 min at room temperature. The samples were centrifuged for 15 min at 4?C with 12,000 g. The upper aqueous phase was transferred to a new vial and 2 ?L 5 M linear acrylamide, 10% volume fraction of 3 M sodium acetate, and an equal volume of 100%
isopropanol were added. Mixtures were vortexed and incubated overnight at -20?C in order to precipitate the RNA. The RNA pellet was collected by 20 min centrifugation at 4°C and 12,000 g. The pellet was washed twice, first with 70% ethanol and afterwards with
96% ethanol, air-dried and dissolved with 100 ?L RNase free water. The RNA sample was further cleaned with the RNeasy Kit according to
manufacturer’s protocol including on-column DNA digestion. RNA quality
check was performed using a NanoDrop ND-100 spectrometer for purity, and the RNA Nano Chip Assay with a 2100 Bioanalyzer was performed in order to examine the integrity of the extracted RNA. Only high quality RNAs (OD 260/OD280>2 and OD260/OD230>1.8) as well as RNA with intact ribosomal peaks were used for microarrays.
(Parameters: Extracted product = total_RNA, Amplification = none)
RNA from the 380 µatm CO2 treatments were pooled. Three labeling reactions were carried out and pooled again afterwards.