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Pipeline publication

[…] icroarrays was performed as described elsewhere [] using an automated hybridization instrument (a-Hyb Hybridization Station, Miltenyi Biotec)., For mRNA profiling, mRNA was amplified using the ╬╝MACS SuperAmp Kit (Miltenyi Biotec) and hybridized on Agilent whole genome arrays., The miRNA microarray design of the miRXplore microarrays (Miltenyi Biotec) has been described in detail previously []. The exact configuration of each microarray is deposited at the National Center for Biotechnology Information Gene Expression Omnibus (GEO; accession number GSE22460). Analysis of the microarrays was done as described before [, ]., For Agilent whole genome arrays, scanned images were analyzed using the Agilent Feature Extraction software (Version 9.1) by which the local background was subtracted. A two-tailed t test was used to determine the signal versus background significance. The significance values were used for filtering of the data set, considering only those signals with p < .01 on at least three of eight arrays (CD133 and CD34 samples) or two of four arrays (Neg samples). After that, the signal intensity data was normalized to the array median and multiplied by the median calculated from all values., For downstream statistical analysis of miRNA and mRNA array data, ratios were log 2 transformed and data imported into the MeV program, which is part of the TM4 package []. Two-dimensional hierarchical clustering was done using Euclidean distance. The statistical analysis of microarrays was performed using significance analysis of microarrays (SAM) with at least 100 permutations per analysis, t test with p < .05 and adjusted Bonferroni correction or t test with p < .01., The miScript PCR System (Qiagen) was used to validate miRNAs detected as differentially expressed on microarrays. The real-time polymerase chain reaction (RT-PCR) reaction and the real-time PCR were carried out according to the manufacturer's protocol. Data were normalized using RNU6B (MS00014000) or RN5S1(MS00007574). PCR reactions were performed in triplicates and carried out in an ABI Prism 7000 SDS Real Time PCR machine (Applied Biosystems, Foster City, CA, http://www.appliedbiosystems.com) and analyzed using ABI Pri […]

Pipeline specifications

Software tools Agilent Feature Extraction, TM4, SAM
Databases GEO