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Sample GSM861296 Query DataSets for GSM861296
Status Public on Aug 08, 2012
Title nemacWT_3 signal
Sample type RNA
 
Source name Nematode (Brugia malayi) elicited peritoneal macrophages from wild type BALB/c mice
Organism Mus musculus
Characteristics tissue: WT peritoneal macrophages
mouse strain: BALB/c
activation status: Nematode (Brugia malayi) 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: slide7.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 10.960938
2729 5.986931
2837 9.307102
1321 82.573009
1429 58.405541
1537 58.500568
4918 12.252274
5026 16.938068
5134 18.377984
3297 6.732529
3405 6.799008
3513 9.956959
2684 7.022587
2792 8.0625
2900 8.075993
689 2.322014
797 6.564774
905 12.294461
1670 31.913921
1778 18.839203

Total number of rows: 5184

Table truncated, full table size 72 Kbytes.




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

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