|
| Status |
Public on Aug 08, 2012 |
| Title |
thioIL4_2 signal |
| Sample type |
RNA |
| |
|
| Source name |
Thioglycollate elicited peritoneal macrophages from IL4 receptor KO BALB/c mice
|
| Organism |
Mus musculus |
| Characteristics |
tissue: IL4 receptor ko peritoneal macrophages
mouse strain: BALB/c
activation status: Thioglycollate elicited
|
| Treatment protocol |
Peritoneal exudate cells were seeded at 5x10^6 cells per well to 6-well cell-culture plates (NUNC) in RPMI, 5% FCS, 2 mM L-glutamine, 0.25 U/mL penicillin and 100 mg/mL streptomycin. After 4 hours incubation at 37oC / 5% CO2 non-adherent cells were washed off and the adherent MΦs detached using a rubber-policeman. Detached cells were > 80% pure as assessed by flow-cytometry (FACS) analysis of their F4/80 and CD11b surface expression.
|
| Growth protocol |
Peritoneal macrophages were elicited 3 days after intraperitoneal thioglycollate injection or 21 days after peritoneal implant of the nematode Brugia malayi. These latter macrophages are termed nematode elicited macrophages (NeMacs).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from macrophages lysed in Qiazol using the miRNEasy-kit (Qiagen) according to the manufacturers instructions. Briefly the RNA from lysed cells was precipitated using Phenol-Chloroform-extraction and the aequeous supernatant purified over a spin-column. RNA-content was determined using a NanoDrop (Thermo Scientific).
|
| Label |
Hy3
|
| Label protocol |
Samples were labelled using Exiqon’s miRCURY LNA array labelling kit (Vedbæk, Denmark): 1ug total RNA was incubated with labelling buffer, the Hy3 dye, labelling enzyme and spike-in microRNAs for 1 hour at 37°C. The enzyme was then heat-inactivated at 65°C for 15 minutes, followed by addition of 2 x hybridisation buffer. The samples were incubated at 95°C for 5 minutes while protected from light. The samples were then briefly spun down and filtered through a 0.45-micron durapore filter (Millipore), and 40 ul sample was loaded to the arrays by capillary force using a cover slip (Erie Scientific).
|
| |
|
| Hybridization protocol |
The arrays were incubated at 60°C for 16 hours in a hybridisation oven.
|
| Scan protocol |
After hybridisation the slides were manually washed for 2 minutes in Buffer A at 60oC, rinsed in Buffer B followed by a 2-minute wash in buffer B at RT and 2 minutes in Buffer C at RT. All buffers were supplied by EXIQON. After washing the slides were dried by centrifugation for 2 minutes at 1000rpm. The slides were immediately scanned using an Agilent G2565BA scanner with a spot size of 10um and 100% PMT gain.
|
| Description |
raw data: slide14.txt (rows 1 to 20821)
|
| Data processing |
Each slide contains two arrays. Two samples were run per slide in separate hybridisation chmabers. Rows 1 to 20820 represent signals for one biological sample and rows 20821 to 41557 represent signals for the second biological sample on the slide. QuantArray software was used to quantify the signals from the Agilent scanner using the fixed circle approach. Background subtracted signal intensities were normalized in R using quantile normalization.
|
| |
|
| Submission date |
Jan 12, 2012 |
| Last update date |
Aug 08, 2012 |
| Contact name |
Iain Gallagher |
| E-mail(s) |
iaingallagher@gmail.com
|
| Phone |
00 44 1786 46 6024
|
| Organization name |
University of Stirling
|
| Department |
Faculty of Health Sciences and Sport
|
| Street address |
Room 4B133 Cottrell Building, University of Stirling, Airthrey Road
|
| City |
Stirling |
| ZIP/Postal code |
FK9 4LA |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL15121 |
| Series (1) |
| GSE35047 |
MicroRNA expression in thioglycollate and alternatively activated |
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