Microarray expression values were analyzed with the Expressionist Analyst Pro v5.2 software (Genedata, Basel, Switzerland). Probe sets were median condensed and linear array-to-array normalization applied by using normalization to a reference set of 48 invariant genes and a reference value of 10.000. The normalized data file contains identifiers from both array designs.
After hybridization the chip was washed and scanned. A 20x SSPE solution was prepared consisting of 175.3 g NaCl, 27.6 g NaH2PO4, 9.4 g EDTA solved in 1 liter water and adjusted to pH 7.4 with NaOH. The hybridization solution was removed and the array was first rinsed then filled with wash solution 1 consisting of 1.8 ml 20x SSPE, 30 µl 10% Tween-20 and 4.17 ml water. The chip was rotated in the oven for 5 min at 45°C. Wash solution 1 was removed and the array was rinsed and filled with wash solution 2 consisting of 0.9 ml 20x SSPE, 30 µl 10% Tween-20 and 5.07 ml water. These steps were repeated with wash solution 3-5. Wash solution 3: 150 µl 20x SSPE, 30 µl 10% Tween-20, 5.82 ml water. Wash solution 4: 1.2 ml 10x PBS, 60 µl 10% Tween-20, 4.74 ml water. Wash solution 5: 1.2 ml 10x PBS, 4.8 ml water. After wash solution 5 the array surface was covered with CombiMatrix imgaging solution and scanned with the Axon GenePix 4200A scanner. After adjusting the focus the array surface was scanned with red channel laser power 30%, PMT gain 300, resolution = 5 µm, lines to average = 3.
Directly after fragmentation ((Ambion #8740, Darmstadt, Germany) of 4 µg Cy5-labeled aRNA, hybridisation was performed (according to CustomArray™ 90K Microarray: Hybridization and Imaging Protocol (PTL020)). The microarray surface was re-hydrated with 120 µl nuclease-free water and sealed with RealTime Folie (MicroAmp™, optical adhesive tape, Applied Biosystems Deutschland GmbH, Darmstadt, Germany). Rehydration was performed for 10 minutes at 65 °C in the hybridisation oven (HB-1000 Hybridizer, Ultra-Violet Products Ltd, Cambridge, UK). After cooling down to room temperature, the water was removed from the hybridisation chamber and immediately filled with 120 µl prehybridisation solution consisting of 60 µl 2x hybridisation solution, 41 µl nuclease-free water, 12 µl 50 x denhardt solution, 6 µl 1% SDS and 1 µl salmon sperm DNA, which was denaturated at 95 °C for 5 minutes prior to use. With insertion of an air bubble and rotation of the microarray chip in the hybridisation oven for 30 minutes at 45 °C, constant distribution of the solution was guaranteed. For actual hybridisation, prehybridisation solution was removed and hybridisation chamber was immediately filled with 120 µl fresh prepared hybridisation solution consisting of 60 µl 2x hybridisation solution, 30 µl deionized formamide, 5 µl 1% SDS, 1 µl denatured salmon sperm DNA and 24 µl labeled and fragmentated aRNA. Insertion of an air bubble ensured equal distribution for 15 hours at 45 °C over the whole microarray surface area. Combimatrix Cy5 microarrays were used up to 4 times, randomly reused between different biological replicates of one treatment to exclude array-specific binding. For this purpose, stripping was performed after imaging like described in the protocol Stripping and Preparation of CombiMatrix 90K Microarrays for Re-hybridization (PTL025) and stripping quality was checked by scanning the chip with scan settings as used for imaging the array.
According to manufacturer’s instructions, 5 µg of aRNA were labeled with Cy5 using the RNA ampULSe-Amplification and Labeling Kit (with Cy5 for CombiMatrix arrays) from Kreatech after in-vitro transcription. After dye removal using KREApure columns, DOL (degree of labeling) was determined with Nanodrop measurement as well. 4 µg of labeled aRNA were used for hybridization.
P. patens gametophores grown for three weeks under standard conditions (see growth protocol). 100 - 150 non-vascular leafs were detached with forceps, placed on Knop minimal medium and incubated at standard conditions.
50-100 mg fresh weight gametophores were snap-frozen in liquid nitrogen immediately after harvesting and disrupted for 90 seconds with a frequency of 30 Hertz using a ball mill (TissueLyser, Qiagen, Hilden, Germany or MM400, Retsch, Haan, Germay). RNA was extracted using RNeasy Plant Minikit from Qiagen with buffer RLT. DNAse digestion was performed directly on the RNeasy spin column membrane using DNAseI from Qiagen according to manufacturer´s protocol. The elution step was performed twice each with 30 µl RNAse free water to obtain a higher RNA yield. The elution volume was reduced by concentration with a SpeedVac (SVC100 and Universal Vacuum System Plus UVS 400A fromSAVANT, Egelsbach, Germany. Vacuum pump DUO5 from Pfeiffer, Asslar, Germany) to a final volume of 15 µl. RNA concentration was measured with the NanoDrop spectrophotometer ND-1000 (peQLab, Erlangen, Germany). RNA integrity was checked by agarose gel electrophoresis and 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA).Amplification of RNA was performed using Amplification und Labeling Kit RNA ampULSe with Cy5 ULS for CombiMatrix Microarraysfrom Kreatech Biotechnology (#GEA-022, Biozym, Hess. Oldendorf, Germany). 0.5 - 1.5 µg RNA were reverse transcribed using T7 Oligo(dT) primer, second strand cDNA synthesis complemented the second strand with DNA polymerase. After cDNA purification, in-vitro transcription reaction was incubated for 4-14 hours at 37°C. The aRNA was again purified and concentrated. Check of quantity and quality of aRNA was carried out as previously described for the RNA.
The wild type strain used was Gransden 2004 (Rensing et al., Science 2008). P. patens gametophores were grown from isolated gametophore stems for three weeks in standard conditions. Standard conditions consisted of a light period from 6 am to 10 pm with average light intensities of 70 µmol * m-2 * s-1. The provided media consisted of Knop minimal medium on 1.2% Agar in 9 cm petri dishes. Knop minimal medium consists of 0.25 g/L KH2PO4, 0.25 g/L KCl, 0.25 g/L MgSO4 * 7 H2O, 1 g/L Ca(NO3)2 * 7 H2O, 12.5 mg/L FeSO4 * 7 H2O, adjusted to pH 5,8 with KOH.