5 fmol of each of 816 synthetic miRNAs were pooled and labelled.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted as described in: Pfeffer, S., Lagos-Quintana, M., and Tuschl, T. (2003). Cloning of small RNA molecules. In Current Protocols in Molecular Biology, F. M. Ausubel, R. Brent, R. E. Kingston , D. D. Moore, J. G. Seidmann, J. A. Smith, and K. Struhl, eds. (New York, John Wiley and Sons), pp. 26.24.21-26.24.18.
Label
Hy3
Label protocol
5 µg total RNA was labelled using a commercial kit (miRCuryTM LNA microRNA Array labeling kit, Exiqon) following the instructions of the manufacturer
Total RNA was extracted as described in: Pfeffer, S., Lagos-Quintana, M., and Tuschl, T. (2003). Cloning of small RNA molecules. In Current Protocols in Molecular Biology, F. M. Ausubel, R. Brent, R. E. Kingston , D. D. Moore, J. G. Seidmann, J. A. Smith, and K. Struhl, eds. (New York, John Wiley and Sons), pp. 26.24.21-26.24.18.
Label
Hy5
Label protocol
5 µg total RNA was labelled using a commercial kit (miRCuryTM LNA microRNA Array labeling kit, Exiqon) following the instructions of the manufacturer
Hybridization protocol
5 µg of respective total RNA were mixed with 2.5 fmol of each of four RNA oligonucleotides reverse complement to miRControl 1 probes and 5 fmol of each of 20 RNA oligonucleotides reverse complement to miRControl 3 probes and subsequently fluorescently labelled by 3’ ligation. Total RNA mix was hybridized in a dual colour approach to microarrays versus a second labelled synthetic miRNA pool. The sy
Scan protocol
Image capture was done with Agilent DNA-Microarray Scanner (Agilent, Santa Clara, CA)
Description
PMID
Data processing
Signal processing and quantification was done with ImaGene software version 5.0 (BioDiscovery, Los Angeles, CA). For each spot, the local signal was measured inside a fixed circle of 230-280 µm diameter, and background was measured outside the circle within specified rings 30 µm distant to the signal and 100 µm wide. Signal and background was taken to be the average of pixels between defined low and high percentages of maximum intensity with percentage parameter settings for low/high being 2/97% for signal and 0/97% for background. Local background was subtracted from the signal to obtain the net signal intensity and the ratio of both fluorescent labels. Subsequently, the mean of the ratios of 4 corresponding spots representing the same miRNA was computed for those spots only which were unflagged (empty spots, poor spots, negative spots) and for which the fluorescent intensity in of the miRNAs derived from the samples of interest was two-fold the mean background value (2bkg dataset). The mean ratios of all probes were normalized to the median of the ratios detected for the spiked 20 synthetic RNA oligonucleotides reverse complement to miRControl 3 probes.
