strain: N402 developmental stage: germinating conidia conidial age: 12 days
Treatment protocol
Control samples of germinating conidia of Aspergillus niger N402 were taken in triplo at 0h (dormant), 2h, and 8h after inoculation in CM. Natamycin treated samples were taken in triplo at 2h and 8h after inoculation in CM either supplemented with 3 µM or 10 µM natamycin. A single experiment comprises three cultures of 300 ml CM with 3 x 109 conidia, shaken at 125 rpm and 25°C. At each time point, 15 ml was taken from each culture and pooled, resulting in 4.5 x 108 conidia per 45 ml. The conidial suspensions were centrifuged at 1100 x g at 5°C for 5 min. Pellets were frozen in liquid nitrogen and kept at -80°C.
Growth protocol
For spore isolation, Aspergillus niger N402 was grown for 12 days at 25 °C on complete medium (CM). Conidia were harvested in ice-cold ACES-buffer (10 mM ACES, 0.02 % Tween-80, pH 6.8). To this end, the colony surface was gently rubbed with a sterile T-spatula and the conidial suspension was filtered through sterile glass wool. Conidia were washed in ice-cold ACES-buffer, resuspended in CM and kept on ice until further processing on the same day.
Extracted molecule
total RNA
Extraction protocol
The conidial pellet was kept frozen in liquid nitrogen and homogenized with the Qiagen Tissuelyser® (2 times 2 min at 30 strokes sec-1) in a stainless steel grinding jar that had been cooled with liquid nitrogen (Qiagen, Venlo, The Netherlands). After homogenizing, 2 ml RLT buffer (supplied with the Qiagen RNeasy® Maxi kit) was added. All the material of the samples taken at 0 h and 2 h, half of the material taken at 4 h and a third of the material taken at 6 h and 8 h were transferred to a 50 ml Greiner tube. RNA was extracted following the protocol of the RNeasy® Maxi kit (Qiagen) with some modifications. 15 ml RLT buffer supplemented with 170 µl ß-mercaptoethanol was added. After centrifugation (3000 x g, 10 min, 4 ºC) the supernatant was mixed thoroughly with 15 ml 70 % ethanol in a 50 ml Greiner tube and transferred to a RNeasy Maxi column. After centrifugation (3000 g, 5 min, 4 °C) the column was washed with 10 ml RW1 and twice with 10 ml RPE buffer (with 2 and 10 min centrifugation, respectively, at 3000 g at 4 °C). This was followed by addition of 800 µl of RNase-free water to elute the RNA. After 2 min the column was centrifuged at 4 °C for 3 min at 3000 g, followed by an additional elution with 800 µl of RNase-free water. The volume of the aqueous RNA solution was reduced to approximately 100-400 µl with a SpeedVac® (Savant DNA 110). Subsequently, 600 µl of 2 x T & C lysis buffer (Epicentre, Landgraaf, The Netherlands) was added and the mixture was kept on ice. After 5 min, 350 µl of MPC Protein Precipitation Reagent (Epicentre, Landgraaf, The Netherlands) was added, thoroughly mixed, and centrifuged (12.000 x g, 10 min, 4 °C) The supernatant was transferred to a clean micro-centrifuge tube and gently mixed with 1000 µl isopropanol. RNA was precipitated at 12.000 x g (10 min, 4 °C) and the pellet was air-dried for 5 min. The pellet was then resuspended in 100 µl RNase-free water. This was followed by addition of 700 µl of RLT buffer (without ß-mercaptoethanol) and 500 µl 96 % ethanol. RNA was further purified using the RNeasy® Mini kit (Qiagen) according to the RNA Cleanup protocol.
Label
biotin
Label protocol
Biotin-labeled antisense cRNA was produced from 2 μg of total RNA with the Eukaryotic One-Cycle Target Labeling kit (Affymetrix; www.affymetrix.com).
Hybridization protocol
Following fragmentation, 10 ug of cRNA was hybridized to Affymetrix A. niger Genome Gene chips (GPL6758). GeneChips were washed and stained in an automated process.
Scan protocol
GeneChips were scanned.
Description
Aniger_T2-10µM-1
Data processing
Absolute values of expression were calculated from the scanned array using the Affymetrix Command Console v1.1 software. Normalization was done using the MAS5.0 algorithm using a baseline correction of the median (the Value '0' was transformed into 12 to enable calculations).
