|
| Status |
Public on May 17, 2011 |
| Title |
single hyphal tip, biological rep4 |
| Sample type |
RNA |
| |
|
| Source name |
exploring hyphae
|
| Organism |
Aspergillus niger |
| Characteristics |
biological rep: 4
tissue: hyphal tip
strain: AR9#2
|
| Treatment protocol |
After removing the Lumox membrane from the sandwiched colony, the mycelium and the underlying PC membrane were cut with a scalpel and part of the periphery of the colony was placed upside down onto a nucleotide and RNAse free glass slide. The PC membrane that was now facing the air was removed and the mycelium was fixed with 70% ethanol and air-dried. The hyphal tips were isolated by laser pressure catapulting (LPC) using the PALM CombiSystem (Carl Zeiss MicroImaging, Germany). This system was equipped with an Axiovert 200M Zeiss inverted microscope (Carl Zeiss, Germany) and a 3CCD color camera (HV-D30, Hitachi Kokusai Electric, Japan). The PALM CombiSystem was operated with PALM RoboSoftware V4.0 (Carl Zeiss MicroImaging, Germany). The autoLPC option was routinely used in combination with a 40x objective. Hyphal material was catapulted into lids of 0.5 ml Eppendorf tubes that contained 50 µl RNAlater (Qiagen, Germany).
|
| Growth protocol |
A. niger Strain AR9#2 was cultured as a sandwiched colony at 30 °C in the light. To this end, the fungus was grown between a perforated polycarbonate (PC) membrane (diameter 76 mm, pore size 0.1 µm; Osmonics, GE Water Technologies, MN) and a Lumox membrane (diameter 76 mm; Greiner Bio-One, Germany) (10). The PC membrane was placed on top of solidified (1.5% agar) minimal medium (MM) (15) containing 25 mM maltose as a carbon source. Freshly harvested spores (1.5 µl of a solution of 0.8% NaCl and 0.005% Tween-80 containing 108 spores ml-1) were placed in the center of the PC membrane. The droplet was allowed to dry, after which the Lumox membrane was placed on top of the PC membrane with its hydrophobic side facing the inoculum.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Hyphal material that was collected in 50 µl RNAlater was transferred to a 2 ml Eppendorf tube by a quick centrifugation step. After snap-freezing in liquid nitrogen, 2 pre-cooled metal bullets (4.76 mm in diameter) were added and samples were ground in a Micro-Dismembrator U (B. Braun Biotech Int.) in a chilled container at 1500 rpm for 60 s. The frozen material was taken up in 250 µl Trizol Reagent (Invitrogen, CA) by vortexing. After removing the metal bullets, 200 µl chloroform was added. After mixing well, samples were centrifuged at 10.000 g for 10 min. The water phase (approximately 200 µl) was mixed with 700 µl RLT from the RNeasy MinElute Cleanup Kit (Qiagen, Germany) to which 143 mM ß-mercaptoethanol was added. RNA was purified following instructions of the manufacturer and was eluted with 12 µl RNAse free water. RNA samples were amplified using the WT-Ovation One-Direct RNA Amplification System (Nugen, CA). The quality and quantity of the cDNA samples were checked using a Bioanalyzer (Agilent Technologies, CA) and a Nanodrop (Nanodrop Technologies, DE), respectively.
|
| Label |
biotin
|
| Label protocol |
Five µg of amplified cDNA was fragmented via combined chemical and enzymatic fragmentation using the protocol of the Encore Biotin Module (Nugen, CA). The fragments were biotin-labeled to the 3-hydroxyl end using the same Module following the instructions of the manufacturer.
|
| |
|
| Hybridization protocol |
The labeled cDNA was hybridized to Affymetrix GeneChip A. niger Genome Arrays. The GeneChip Hybridization, Wash and Stain Kit (Affymetrix, CA) was used for the hybridizations according to the protocol of the manufacturer with the modification that the hybridization cocktail was prepared according to the Encore Biotin Module and that the hybridization time was extended to 40 h.
|
| Scan protocol |
GeneChips were scanned using the Agilent GCS3000 HR GeneChip Scanner and Affymetrix GeneChip Command Console Software (AGCC v1.1)
|
| Description |
Gene expression data from a single hyphal tip collected from periphery material of a single colony
101301-4
|
| Data processing |
The data were analyzed with the R statistical programming language (R-2.11.1) and the bioconductor affy package. Present/absent call were generated with the MAS5 algorithm and summarized probesets values using the RMA algorithm
|
| |
|
| Submission date |
Nov 19, 2010 |
| Last update date |
May 17, 2011 |
| Contact name |
Oskar Bruning |
| Organization name |
University of Amsterdam
|
| Department |
SILS
|
| Lab |
MicroArray Department
|
| Street address |
Science Park 904
|
| City |
Amsterdam |
| ZIP/Postal code |
1098 XH |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL6758 |
| Series (1) |
| GSE25497 |
Single tip transcriptomics of neighboring hyphae of Aspergillus niger |
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