|
| Status |
Public on Jun 15, 2009 |
| Title |
Lung_CBA OVA v Lung CBA PBS rep2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
CBA OVA treatment
|
| Organism |
Mus musculus |
| Characteristics |
strain: CBA
tissue: lung
treatment: ovalbumin
|
| Treatment protocol |
Lung tissue was recovered 72 h after OVA or PBS challenge. Both lungs were placed in 2 ml of Trizol and homogenized. RNA was extracted according to the manufacturer’s instructions.
|
| Growth protocol |
Mice were immunized by intraperitoneal injection of 10 ug OVA mixed with 1 mg of aluminium hydroxide gel in 50 uL of endotoxin-free (ef) PBS, on each of days 0 and 12. Using a clinical nebulizer, mice were then challenged with aerosols of 1% OVA in efPBS or with efPBS alone, for either 20 min on each of days 28 and 29. Mice were euthanized by CO2 inhalation on day 30 or 32.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using Trizol according to the manufacturer’s instructions. The extracted RNA was purified using an RNEasy column (Qiagen) according to the manufacturer’s instructions.
|
| Label |
Cy3
|
| Label protocol |
cDNA was made from total RNA (>20μg) using Superscript II, anchored polyT(V)N (2μg/μL) and an aminoallyl dNTP mix. The RNA was hydrolysed, the cDNA purified then coupled to Cy3 or Cy5 NHS esters (detailed protocols can be obtained from www.microarray.adelaide.edu.au)
|
| |
|
| Channel 2 |
| Source name |
CBA PBS treatment
|
| Organism |
Mus musculus |
| Characteristics |
strain: CBA
tissue: lung
treatment: saline
|
| Treatment protocol |
Lung tissue was recovered 72 h after OVA or PBS challenge. Both lungs were placed in 2 ml of Trizol and homogenized. RNA was extracted according to the manufacturer’s instructions.
|
| Growth protocol |
Mice were immunized by intraperitoneal injection of 10 ug OVA mixed with 1 mg of aluminium hydroxide gel in 50 uL of endotoxin-free (ef) PBS, on each of days 0 and 12. Using a clinical nebulizer, mice were then challenged with aerosols of 1% OVA in efPBS or with efPBS alone, for either 20 min on each of days 28 and 29. Mice were euthanized by CO2 inhalation on day 30 or 32.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using Trizol according to the manufacturer’s instructions. The extracted RNA was purified using an RNEasy column (Qiagen) according to the manufacturer’s instructions.
|
| Label |
Cy5
|
| Label protocol |
cDNA was made from total RNA (>20μg) using Superscript II, anchored polyT(V)N (2μg/μL) and an aminoallyl dNTP mix. The RNA was hydrolysed, the cDNA purified then coupled to Cy3 or Cy5 NHS esters (detailed protocols can be obtained from www.microarray.adelaide.edu.au)
|
| |
|
| |
| Hybridization protocol |
Labeled cDNA, yeast tRNA, poly A and Cot-1 DNA were mixed with 16uL of formamide and 16uL of 6.25 X SSC. The mixture was heated to 100C for 3min. and applied to the centre of the microarray. Hybridisation occurred at 42C overnight in a humidified chamber. The array was then washed in 0.5 X SSC containing 0.01% SDS for 5 min., 0.5 X SSC for 5 min then 0.2 X SSC for 3 min. then dried by centrifugation (detailed protocols can be obtained from www.microarray.adelaide.edu.au).
|
| Scan protocol |
Microarrays were scanned using an Axon 4000B microarray scanner.
|
| Description |
n/a
|
| Data processing |
Single channel .tif images of microarray slides were analysed using SPOT software. The data was normalised and anlaysed using Lima GUI.
|
| |
|
| Submission date |
Mar 23, 2009 |
| Last update date |
Jun 15, 2009 |
| Contact name |
Ashley Connolly |
| Organization name |
University of Queensland
|
| Street address |
St Lucia
|
| City |
Brisbane |
| ZIP/Postal code |
4068 |
| Country |
Australia |
| |
|
| Platform ID |
GPL3069 |
| Series (2) |
| GSE15345 |
Expression of survivin in lung eosinophils is associated with pathology in a mouse model of allergic asthma 1 |
| GSE15429 |
Expression of survivin in lung eosinophils is associated with pathology in a mouse model of allergic asthma |
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