Common garden experiment in wet lab. Each full-sib family of eggs was divided into two batches, which were incubated at 5 and 8 degrees C in hatching troughs with circulated water, biological filter and temperature control. Each family was placed in separate compartments of hatching boxes, but all families and individuals experienced similar conditions.
Extracted molecule
total RNA
Extraction protocol
RNA was extracted from whole individuals on ice using the RNA Pure Link Minikit (Invitrogen, Paisley, UK). Each fry was homogenized individually in 0.8ml lysis buffer containing 1% 2-mercaptoethanol using a Tissuelyzer (QIAGEN, Hilden, Germany). Samples were then centrifuged at 2600 g for 5 min and 0.6 ml of the supernatant was transferred to a new 2 ml tube, 0.6 ml Trizol and 0.6 ml chloroform was added, mixed and centrifuged at 13,000 g at 4¡C for 15 min. Then, 800 µl of the upper phase was transferred to a new tube and RNA was extracted according to the manufacturer's recommendations. After RNA extraction, 1 µl of RNase inhibitor (20 units/µl, Ambion, Invitrogen, Paisley, UK) was added to prevent RNA degradation and samples were DNase treated with DNase I, amplification grade (1 unit/µl) (Invitrogen, Paisley, UK) following the manufacturer's recommendation. RNA quantity and integrity was checked on a Nanodrop instrument (ND-1000; Thermo Fisher Scientific Inc., Wilmington, Delaware, USA) and on an Experion automated electrophoresis station using RNA HighSens chips (BIORAD, Copenhagen, Denmark).
Label
Cy3
Label protocol
Cy5 and Cy3 dyes of the Array50 kit (Genisphere) following the procedures at http://web.uvic.ca/cbr/grasp/microarray/array.html (Genisphere Array 50 Protocol Revised version 5), using 15microgram of RNA per reverse transcription reaction and sunbsequently OPTION 1 Ð concentration of cDNA using Millipore Microcon YM-30 concentrators.
Scanned using a ScanArray scanner (PerkinElmer) at a resolution of 10μm and 90% laser power, manually equilibrating intensity between dyes for each slide using the photomultiplier tube settings.
Description
T5_Norring
Data processing
Spots were localized and quantified with the QuantArray 3.0 software (Perkin- Elmer Life Sciences), using the histogram quantification method and keeping the mean value of intensity for each spot. Local background was removed and the data from bad spots were manually excluded from the data set. For each of the two dyes on each array, spots with signal intensities lower than the mean intensity of the empty spots plus twice their standard deviation were flagged as non-expressed. Spots that had no non-expressed flag in at least one of the six studied groups (3 rivers and 2 temperatures) were kept.
