TRIzol reagent (Invitrogen, Carlsbad, CA) (500 µl) was added to dry cell pellets, which were vigorously pipetted. After incubation at room temperature (5 minutes), chloroform (100 µl) was added, and the sample was briefly vortexed and again incubated at room temperature (5 minutes). After centrifugation (15 minutes, 12,000g, 4°C), the upper aqueous phase of samples was transferred to a new RNase-free microcentrifuge tube containing 250 µl of isopropanol. Following pulse-vortexing and incubation at room temperature (5 minutes), total RNA was precipitated (20 minutes, 14,000g, 4°C). Following removal of the supernatant, the RNA pellet was washed in 500 µl of 70% ethanol (30 minutes, 14,000g, 4°C), RNA pellets were allowed to air-dry (10 minutes) and were resuspended in 20 µl of RNase-free water. Total RNA samples were evaluated spectrophotometrically using a Nanodrop spectrophotometer. A260 values were used to quantify the samples, and A260/A280 ratios were used to determine relative purity; generally, RNA samples with A260/A280 ≥1.6 were routed to analysis.
Label
Cy3
Label protocol
Purified miRNA was hybridized to the Human miRNA Microarray platform (Agilent, Santa Clara, CA) following the Agilent version 2.0 protocol (June 2008) explicitly for target labeling, hybridization, washing, scanning, and image analysis.
Hybridization protocol
Purified miRNA was hybridized to the Human miRNA Microarray platform (Agilent, Santa Clara, CA) following the Agilent version 2.0 protocol (June 2008) explicitly for target labeling, hybridization, washing, scanning, and image analysis.
Scan protocol
Purified miRNA was hybridized to the Human miRNA Microarray platform (Agilent, Santa Clara, CA) following the Agilent version 2.0 protocol (June 2008) explicitly for target labeling, hybridization, washing, scanning, and image analysis.
Description
miRNA expression of CD138 selected plasma cells
Data processing
Purified miRNA was hybridized to the Human miRNA Microarray platform (Agilent, Santa Clara, CA) following the Agilent version 2.0 protocol (June 2008) explicitly for target labeling, hybridization, washing, scanning, and image analysis. Total gene signal from GeneView data files, which were extracted using default miRNA settings in Agilent Feature Extraction (v9.5.x), was used as signal for miRNAs. We took advantage of spiked-in controls spotted in Agilent's platform, which were external references and served as microarray controls in the hybridization protocol. We averaged intensities of positive controls on each array and then normalized miRNA signals by equalizing these averages among all arrays.
