Microarrays were manufactured by Miltenyi Biotec. Briefly, 1.2 nl of 10 µM HPLC purified, 5’ amino-modified DNA oligonucleotides (Metabion) were spotted on aminopropyltrimethoxysilane / phenylendiisothiocyanate coated glass slides in quadruplicates using a customized piezoelectric spotter. Microarrays were manufactured by Miltenyi Biotec. Briefly, 1.2 nl of 10 µM HPLC purified, 5’ amino-modified DNA oligonucleotides (Metabion) were spotted on aminopropyltrimethoxysilane / phenylendiisothiocyanate coated glass slides in quadruplicates using a customized piezoelectric spotter.
Description
Each microarray contained a total of 5308 DNA oligonucleotides with a sequence being reverse complement to the respective mature miRNA or control RNA, representing a total of 2012 miRNA sequences and 97 controls with all probes spotted in quadruplicates. The 2012 miRNA sequences covered sequence verified miRNAs of the species human (849) mouse (616) and rat (426) as well as viral miRNAs (121) as deposited in the miRBase sequence database version 11.0. The 97 positive and negative control probes were chosen as follows. Three sets of controls, miRControl 1 (four sequences used to assess efficiency and sensitivity of labelling), miRControl 2 (single sequence used to assess efficiency of small RNA enrichment) and miRControl 3 (43 sequences used to normalize signal intensities across different samples and arrays) were artificial sequences chosen not be present in any of the human, mouse, rat or viral genome. A fourth set of positive controls presented 36 positive small but non-miRNA sequences including probes e.g. for 5.1 sRNA, tRNAs, and U6 RNAs. A fifth set of oligonucleotides included 13 negative controls like different single or double mismatch, antisense, or scrambled miRNA sequences. Microarrays were manufactured by Miltenyi Biotec. Briefly, 1.2 nl of 10 µM HPLC purified, 5’ amino-modified DNA oligonucleotides (Metabion) were spotted on aminopropyltrimethoxysilane / phenylendiisothiocyanate coated glass slides in quadruplicates using a customized piezoelectric spotter. Each microarray contained a total of 5308 DNA oligonucleotides with a sequence being reverse complement to the respective mature miRNA or control RNA, representing a total of 2012 miRNA sequences and 97 controls with all probes spotted in quadruplicates. The 2012 miRNA sequences covered sequence verified miRNAs of the species human (849) mouse (616) and rat (426) as well as viral miRNAs (121) as deposited in the miRBase sequence database version 11.0. The 97 positive and negative control probes were chosen as follows. Three sets of controls, miRControl 1 (four sequences used to assess efficiency and sensitivity of labelling), miRControl 2 (single sequence used to assess efficiency of small RNA enrichment) and miRControl 3 (43 sequences used to normalize signal intensities across different samples and arrays) were artificial sequences chosen not be present in any of the human, mouse, rat or viral genome. A fourth set of positive controls presented 36 positive small but non-miRNA sequences including probes e.g. for 5.1 sRNA, tRNAs, and U6 RNAs. A fifth set of oligonucleotides included 13 negative controls like different single or double mismatch, antisense, or scrambled miRNA sequences. Microarrays were manufactured by Miltenyi Biotec. Briefly, 1.2 nl of 10 µM HPLC purified, 5’ amino-modified DNA oligonucleotides (Metabion) were spotted on aminopropyltrimethoxysilane / phenylendiisothiocyanate coated glass slides in quadruplicates using a customized piezoelectric spotter.
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