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| Status |
Public on Sep 27, 2010 |
| Title |
Elf5+/+ mammary gland, 10.5 days post coitus, sample 3 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Elf5 WT 10.5dpc
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
genotype: Elf5+/+
tissue: mammary gland
age: 10.5 days post coitus
|
| Treatment protocol |
Mice were kept under regular 12hr light/dark cycles, and fed ad libitum.
|
| Growth protocol |
Mice were mated, and the day of vaginal plug detection deemed 0.5 days post coitus (dpc). Mice were humanely culled on the appropriate day of pregnancy and the abdominal mammary glands from two mice pooled.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted from the mammary glands using the Rneasy maxi kit (Qiagen) according to the manufacturer's instructions.
|
| Label |
Alexa 532
|
| Label protocol |
The purified cDNA was dried under reduced pressure, and then dissolved in 9 µL of 0.1 M NaHCO3 (pH 9.0). The mixture was added to a Cy dye aliquot (One Cy dye sachet (Amersham) is dissolved in 73 µL of anhydrous DMSO, and then 4.5 µL aliquots are distributed into sealable tubes. Each aliquot is dried under reduced pressure, sealed, and then stored in a dessicator at –20°C), mixed, and then left to incubate at room temperature for 60 mins in the dark. The labelled cDNA was mixed with 41 µL of Milli Q water, and then purified using a QIAquick PCR purification kit (Qiagen). The sample was eluted into a clean tube with 90 µL of Milli Q water. The purified labelled cDNA samples appear purple after being dried under reduced pressure.
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| |
|
| Channel 2 |
| Source name |
Total RNA from pooled whole mouse embryos e17.5
|
| Organism |
Mus musculus |
| Characteristics |
genotype: wildtype
tissue: whole body
development stage: embryo
age: 17.5 days post coitus
sample type: reference
|
| Treatment protocol |
Mice were kept under regular 12hr light/dark cycles, and fed ad libitum.
|
| Growth protocol |
Mice were mated, and the day of vaginal plug detection deemed 0.5 days post coitus (dpc). Mice were humanely culled on the appropriate day of pregnancy and the abdominal mammary glands from two mice pooled.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted from the mammary glands using the Rneasy maxi kit (Qiagen) according to the manufacturer's instructions.
|
| Label |
Alexa 635
|
| Label protocol |
The purified cDNA was dried under reduced pressure, and then dissolved in 9 µL of 0.1 M NaHCO3 (pH 9.0). The mixture was added to a Cy dye aliquot (One Cy dye sachet (Amersham) is dissolved in 73 µL of anhydrous DMSO, and then 4.5 µL aliquots are distributed into sealable tubes. Each aliquot is dried under reduced pressure, sealed, and then stored in a dessicator at –20°C), mixed, and then left to incubate at room temperature for 60 mins in the dark. The labelled cDNA was mixed with 41 µL of Milli Q water, and then purified using a QIAquick PCR purification kit (Qiagen). The sample was eluted into a clean tube with 90 µL of Milli Q water. The purified labelled cDNA samples appear purple after being dried under reduced pressure.
|
| |
|
| |
| Hybridization protocol |
Microarray slides were immersed in 50 ml of hot Milli-Q water (80-95°C) and agitated gently for 5 mins prior to hybridisation. The labelled cDNA was mixed with 0.64 μl of 25 mg/ml yeast tRNA, 4 μl of 2 mg/ml poly A and 20 μl of 1 mg/ml Cot1 DNA. This mix was then dried under reduced pressure, and then dissolved in 16 μl of formamide and 6.25 x SSC. The mixture was then heated to 100°C for 3 mins, transferred directly to ice before adding 0.5 μl of 10% SDS. The solution was then applied to the centre of the cover slip and the array slide was lowered onto the cover slip. The slide was then incubated at 42°C overnight in a humidified chamber. The array slide was then immersed in a solution of 0.5 X SSC and 0.01% SDS until the coverslip detached from the slide. After discarding the coverslip, the slide was washed in 0.5 X SSC and 0.1% SDS for 5 mins, and then 0.5 X SSC for 5 mins and then 0.2 X SSC for 3 mins. The slide was then dried in a centrifuge at 750 rpm for 5 mins and stored in the dark prior to scanning.
|
| Scan protocol |
Microarray slides were scanned using an Agilent G2565BA DNA microarray scanner. Images were analysed with DigitalGENOME (Molecularware).
|
| Description |
10.5 WT 3
Biological replicate 3 of 3.
|
| Data processing |
LOWESS normalised. Prior to analysis, the following filtering steps were applied: 1) only genes with a mean reference (embryo) intensity equal to or above the average reference intensity (average for all the spots in the reference channel on that slide) in at least 6 of the 10 conditions were included; and 2) the list of genes was also filtered on fold change, where genes must (in at least 1 of the 10 comparisons) have normalised data values that are greater or less than those in the other 9 conditions by a factor of 2 fold, i.e. the expression of the gene must change at one or more time points. After these filters were applied, the number of genes on our list was a more manageable 2325. Genespring GX 7.3 (Agilent) software was used for all data processing.
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| |
|
| Submission date |
Aug 31, 2010 |
| Last update date |
Sep 07, 2010 |
| Contact name |
Renee Rogers |
| E-mail(s) |
r.rogers@garvan.org.au
|
| Organization name |
Garvan Institute of Medical Research
|
| Street address |
384 Victoria Street
|
| City |
Darlinghurst |
| State/province |
NSW |
| ZIP/Postal code |
2010 |
| Country |
Australia |
| |
|
| Platform ID |
GPL10871 |
| Series (1) |
| GSE23373 |
Transcript Profiling of Elf5+/- Mammary Glands During Pregnancy Identifies Novel Targets of Elf5 |
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