Clontech's Total RNAs are prepared by the methods referenced in the following: Chomczynski & Sacchi (1987) Anal. Biochem. 162:156-159 Guanidinium thiocyanate method in Maniatis, Sambrook, J. et al (1989) Molecular Cloning: A Laboratory Manual, 2nd edition
Label
Digoxigenin-UTP
Label protocol
Digoxigenin-UTP-labeled cRNA was generated and linearly amplified from 1 µg of total RNA with three replicates per tissue, using the Applied Biosystems Chemiluminescent RT-IVT Labeling Kit v 2.0 and kit protocol.
Hybridization protocol
Microarray hybridization, chemiluminescence detection, image acquisition, and analysis were performed on the on the Applied Biosystems 1700 Chemiluminescent Microarray Analyzer using the Applied Biosystems Chemiluminescence Detection Kit and kit protocol. Each microarray was pre-hybridized at 55oC for 1 hr in hybridization buffer with a blocking reagent. Next, 15 µg of labeled cRNA targets were fragmented by incubating them at 60oC for 30 min with fragmentation buffer and the internal control target (a 24-mer oligo labeled with the LIZ® fluorescent dye), and then hybridized to each microarray in a 1.5-mL volume at 55oC for 16 hr. After hybridization the arrays were washed with hybridization wash buffer and chemiluminescence rinse buffer. Enhanced chemiluminescent signals were generated by incubating the arrays with anti-digoxigenin-alkaline phosphatase and chemiluminescence enhancing solution, followed by chemiluminescence substrate.
Scan protocol
Images from each microarray were collected on the Applied Biosystems 1700 Microarray Analyzer. The images were auto-gridded and the chemiluminescent signals were quantified, corrected for background and spot quality, and spatially normalized.
Description
Human PBLs replicate 1 of 3
Data processing
Images were auto-gridded and the chemiluminescent signals were quantified, corrected for background, and finally, spot- and spatially-normalized using the Applied Biosystems 1700 Chemiluminescent Microarray Analyzer software. The raw data was globally normalized using quantle normalization.
Signal-to-noise ratio. Used for probe detectability. A probe with S/N >=3 is generally considered detected. This threshold can be changed for a given experiment.
FLAG
Quality flag of the feature spot. P indicates good spot, F indicates that the spot may have quantification issues
