All treatments done in vivo. Morphine implanted subcuteniously, LPS administered as intraperitoneal injection.
Growth protocol
Macrophages harvested from mice (primary source)
Extracted molecule
total RNA
Extraction protocol
Trizol® reagent (Invitrogen) was used to extract total RNA from the cells.
Label
DY547
Label protocol
Total RNA was ligated to a synthetic linker, 5’-pCU-DY547-3’. Reference DNA oligonucleotides (Invitrogen) complementary to a subset of mammalian miRNAs were combined and labeled with a ULYSIS® Alexa Fluor® 647 kit.
Hybridization protocol
A mixture of labeled RNA and DNA was hybridized to a miRNA microarray (Thomson et al), inside a MAUI mixer in a MAUI hybridization system (BioMicro® System, Inc, Salt Lake City, UT, USA). Thomson, J.M., Parker, J., Perou, C.M., and S.M. Hammond. 2004. A custom microarray platform for analysis of microRNA gene expression. Nat. Methods 1: 47-53. Epub 2004 Sep 29.
Scan protocol
Slides were scanned with a ScanArray 5000 machine (Perkin Elmer®, Waltham, MA, USA).
Data processing
BlueFuse (BlueGenome, Cambridge, UK) was used to quantify pixel intensities. GeneSpring GX 7.3.1 (Agilent Technologies) was used for data normalization.
