Total RNA was extracted with TRIZOL reagent (Invitrogen, Carlsbad, CA) and the low-molecular-weight RNA was isolated by using the Ambion miRNA Isolation Kit
Label
Cy3
Label protocol
4 ug of low-molecular-weight RNA was labeled with 500 ng of 5'-phosphate-cytidyl-uridyl-cy3-3' (Dharmacon, Lafayette, CO) with 2 units T4 RNA ligase (New England Biolabs, Ipswich, MA). The labeling reaction was performed at 4oC for 2 h. Labeled RNA was precipitated with 0.3 M sodium acetate and 2.5 volumes ethanol and after washing with ethanol and drying was resuspended in 15 ¦Ìl of hybridization buffer containing 3¡ÁSSC, 0.2% SDS and 15% formamide.
Hybridization protocol
Hybridization was performed at 42oC under LifterSlipTM (Erie, Portsmouth, NH) in a hybridization chamber which was placed in a 3D-tilting agitator BioMixerTM II (CapitalBio) to provide continuous mixing of the hybridization buffer that results in more uniform hybridization across the entire slide surface, preventing edge effects and giving improved signal intensity, the efficiency of which has been demonstrated with our genome-wide mRNA expression profiling (Patterson et al., Nat Biotechnol. 2006, 24:1140-1150).The array was then washed with two consecutive washing solutions of 0.2% SDS, 2¡ÁSSC at 42¡ãC for 5 min, and 0.2% SSC for 5 min at room temperature
Scan protocol
Arrays were scanned with a LuxScanTM 10K-A laser confocal scanner and the images obtained were then analyzed using LuxScan 3.0TM software (both from CapitalBio, Beijing, China)
Description
the microRNA expression in the tissue
Data processing
After average values of the replicate spots of each miRNA were background subtracted, faint spots were filtered out when expression signal were lower than 800 in all samples.
