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Sample GSM861291 Query DataSets for GSM861291
Status Public on Aug 08, 2012
Title thioIL4_1 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: slide12.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

Data table header descriptions
ID_REF
VALUE Quantile normalised background subtracted values

Data table
ID_REF VALUE
2621 17.253693
2729 13.245453
2837 16.79858
1321 240.305539
1429 247.603695
1537 174.923294
4918 45.262787
5026 57.246025
5134 29.877274
3297 11.944462
3405 6.997726
3513 13.00341
2684 9.588352
2792 23.029118
2900 12.919033
689 10.714348
797 15.052555
905 11.272728
1670 24.274715
1778 36.637929

Total number of rows: 5184

Table truncated, full table size 74 Kbytes.




Supplementary data files not provided
Raw data are available on Series record
Processed data included within Sample table

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