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| Status |
Public on Dec 31, 2011 |
| Title |
Slide#6_12980415 |
| Sample type |
RNA |
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| Channel 1 |
| Source name |
5-AC 16hr rep4
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| Organism |
Aspergillus parasiticus |
| Characteristics |
tissue: mycelia
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| Treatment protocol |
A. parasiticus SRRC 143 (or SU-1); ATCC # 56775) was used as the wild-type strain. Non-aflatoxigenic A. parasiticus clones derived from SU-1 were prepared by serial mycelial transfer as previously described [Kale et al. 1996] or by 5-AC treatment (1 mM in A&M medium for 36 h) as described for A. nidulans [Tamame et al., 1983].
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| Growth protocol |
For wild-type A. parasiticus (WT), an A. parasiticus 5-AC mutant, and an A. parasiticus serial transfer mutant (serial transfer), fresh conidia were inoculated into 200 ml A&M liquid medium (105 spores/ml) and incubated at 29°C with constant shaking (150 rpm). Mycelia were collected from clones after 16 and 24 hours from cultures. A total of six replicates were performed for each condition, with each replicate consisting of a total of 4 separate cultures. The four cultures were combined and 20 ml of the resultant supernatant was isolated for aflatoxin determination. The combined mycelia was flash frozen in liquid nitrogen in a pre-chilled mortar and pestle and ground to a fine powder before storage at -80°C
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| Extracted molecule |
total RNA |
| Extraction protocol |
Mycelia were collected from clones after 16 and 24 hours from cultures. A total of six replicates were performed for each condition, with each replicate consisting of a total of 4 separate cultures. The four cultures were combined and 20 ml of the resultant supernatant was isolated for aflatoxin determination. The combined mycelia was flash frozen in liquid nitrogen in a pre-chilled mortar and pestle and ground to a fine powder before storage at -80°C. RNA was isolated from 100 mg ground mycelia by suspending in 1 ml of Puresol (Biorad). After treatment with RNase-free DNaseI (Aurum Total RNA fatty and fibrous tissue kit, Biorad), the resulting RNA was converted to cDNA and labeled as previously described (Price et al., 2005).
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| Label |
Cy3
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| Label protocol |
RNA was converted to cDNA and labeled using a protocol modified from Chhabra, et al., 2003. J Biol Chem 278:7540 ( described (Price et al., 2005 Fungal Genet. Biol. 42:506-518.)
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| Channel 2 |
| Source name |
WT 16hr rep3
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| Organism |
Aspergillus parasiticus |
| Characteristics |
tissue: mycelia
|
| Treatment protocol |
A. parasiticus SRRC 143 (or SU-1); ATCC # 56775) was used as the wild-type strain. Non-aflatoxigenic A. parasiticus clones derived from SU-1 were prepared by serial mycelial transfer as previously described [Kale et al. 1996] or by 5-AC treatment (1 mM in A&M medium for 36 h) as described for A. nidulans [Tamame et al., 1983].
|
| Growth protocol |
For wild-type A. parasiticus (WT), an A. parasiticus 5-AC mutant, and an A. parasiticus serial transfer mutant (serial transfer), fresh conidia were inoculated into 200 ml A&M liquid medium (105 spores/ml) and incubated at 29°C with constant shaking (150 rpm). Mycelia were collected from clones after 16 and 24 hours from cultures. A total of six replicates were performed for each condition, with each replicate consisting of a total of 4 separate cultures. The four cultures were combined and 20 ml of the resultant supernatant was isolated for aflatoxin determination. The combined mycelia was flash frozen in liquid nitrogen in a pre-chilled mortar and pestle and ground to a fine powder before storage at -80°C
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Mycelia were collected from clones after 16 and 24 hours from cultures. A total of six replicates were performed for each condition, with each replicate consisting of a total of 4 separate cultures. The four cultures were combined and 20 ml of the resultant supernatant was isolated for aflatoxin determination. The combined mycelia was flash frozen in liquid nitrogen in a pre-chilled mortar and pestle and ground to a fine powder before storage at -80°C. RNA was isolated from 100 mg ground mycelia by suspending in 1 ml of Puresol (Biorad). After treatment with RNase-free DNaseI (Aurum Total RNA fatty and fibrous tissue kit, Biorad), the resulting RNA was converted to cDNA and labeled as previously described (Price et al., 2005).
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| Label |
Cy5
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| Label protocol |
RNA was converted to cDNA and labeled using a protocol modified from Chhabra, et al., 2003. J Biol Chem 278:7540 ( described (Price et al., 2005 Fungal Genet. Biol. 42:506-518.)
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| Hybridization protocol |
Hybridization were performed using a protocol modified from Chhabra, et al., 2003. J Biol Chem 278:7540 ( described (Price et al., 2005 Fungal Genet. Biol. 42:506-518.)
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| Scan protocol |
Hybridized slides were scanned using a ScanArray5000XL (GSI Lumonics, Packard Biochip, Packard BioScience Company, Billerica, MA) and the independent TIFF images from each channel were analyzed using TIGR Spotfinder (http://www.tm4.org/spotfinder.html) software program.
When the slides were printed the columns were slightly off. So when we scanned the arrays we had to do each one twice and then merge the data when we were handling the statistics. Because of this the raw files (.tav) and the processed files are split into two files for each slide (cols 1-13 and 14-24).
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| Data processing |
The hybridized slides were scanned and analyzed for statistically significant different expression levels as previously described (Chang, P.K., Wilkinson, J.R., Horn, B.W., Yu, J., Bhatnagar, D., Cleveland, T.E. Genes differentially expressed by Aspergillus flavus strains after loss of aflatoxin production by serial transfers. Appl Microbiol Biotechnol. 2007, 77, 917-925). Processed data is linked as supplementary file to GSE30756.
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| Submission date |
Jul 18, 2011 |
| Last update date |
Dec 31, 2011 |
| Contact name |
Jeff Wilkinson |
| Organization name |
none
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| Street address |
635 Parson
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| City |
Beaumont |
| State/province |
tx |
| ZIP/Postal code |
77706 |
| Country |
USA |
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| Platform ID |
GPL2122 |
| Series (1) |
| GSE30756 |
Microarray analysis reveals novel regulatory genes associated with the non-aflatoxigenic Aspergillus parasiticus mutants obtained by 5-azacytosine treatment or serial mycelial transfer |
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| Supplementary file |
Size |
Download |
File type/resource |
| GSM763047_Slide_6_12980415_col_14_24.tav.gz |
623.4 Kb |
(ftp)(http) |
TAV |
| GSM763047_Slide_6_12980415_col_1_13.tav.gz |
695.6 Kb |
(ftp)(http) |
TAV |
| Processed data are available on Series record |
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