7-day old sandwiched colonies grown on xylose were used for RNA isolation. Distinct zones of the mycelium (zone 1, 3, and 5) harvested form the colony. Zone 1 represents the youngest or central part of the colony. Zone 5 represents the oldest or peripheral part of the colony, whereas zone 3 represents the intermediate zone.
Extracted molecule
total RNA
Extraction protocol
Mycelium of zones was harvested from three colonies, frozen with liquid nitrogen, and ground with a TissueLyser (Qiagen, Venlo, The Netherlands) in a 2 ml Eppendorf tube with two metal balls (4.76 in diameter) for 1 min at 25 Hz. The frozen material was taken up in 1 ml TRIzol reagent (Invitrogen, Life technologies, Bleiswijk, Netherlands) by vortexing. Samples were incubated for 2 min after mixing with 200 µl chloroform. This was followed by centrifugation at 10000 g for 10 min. RNA was purified using an RNA clean up column (Machery Nagel, Düren, Germany), after addition of 1 volume 70 % EtOH to the water phase. To this end, the sample was loaded on the column, which was centrifuged for 30 sec at 10000 rpm. This was followed by addition of 600 µl RA3 buffer (provided by the RNA-clean up kit). After 2 min of centrifugation at 10000 g, 250 µl RNA3 was added, followed by another 2 min centrifugation at 10000 g. RNA was eluted after 10 min incubation in two steps with 40 µl and 50 µl RNAse free water.
Label
Biotin
Label protocol
The Affymetrix (#901229) 3’ IVT-Express Labeling Kit was used to synthesize Biotin-labeled cRNA. From each RNA sample 100 ng was used as input for the labeling reactions. For the second labeling reaction, 500 ng RNA was used.
Hybridization protocol
15 µg cRNA was fragmented and half of it was used for hybridization on Affymetrix A. niger GeneChips. (Affymetrix #900720)
Scan protocol
Absolute values of expression were calculated from the scanned array using the Affymetrix Command Console v1 software.
Data processing
Genedata Expressionist and Genedata Analyst were used for normalization and statistical analysis of the arrays (Genedata, Basel, Switzerland). The arrays were condensed with the RMA algorithm and normalized on the quantile.
