One hundred nanograms of total RNA from cells at different experimental conditions were treated following the miRNA microarray protocol (Agilent Technologies, Placerville, CA). Briefly, RNA was dephosphorylated and denatured, followed by a ligation and labeling step. Samples were hybridized to Human miRNA Microarray 8 × 60K glass arrays from the Sanger miRBase database release 16 (2006 human miRNAs represented, Agilent Technologies). After hybridization, slides were washed following the Agilent procedure and scanned with the dual-laser Agilent Scanner version C (G2505C, Agilent Technologies). Images were analyzed using the Feature Extraction software v10.7. Raw data elaboration was carried out with Bioconductor (www.bioconductor.org ),32 (link) using R statistical language. The LIMMA package was then used to identify differentially expressed miRNAs between the different experimental conditions. The empirical Bayes method was used to compute a moderated t-statistics.33 miRNAs with a log base two-fold change (logFC) greater than +0.58 or lower than −0.58 were considered as differentially expressed.
Agilent scanner version c
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The Agilent Scanner version C is a high-performance imaging device designed for laboratory and research applications. It captures digital images of samples or specimens with precision and clarity. The scanner's core function is to digitize and create electronic images from physical materials for further analysis and documentation.
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2 protocols using agilent scanner version c
1
miRNA Microarray Protocol for Differential Expression
2
Transcriptome Analysis of DHSF-BR16 and MCF-7 Cells
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Transcriptome analysis was performed on RNA samples of DHSF-BR16 and MCF-7 cells obtained from three independent pellets as reported above .
Gene expression profiling was carried out using the one-color labelling method: labelling, hybridization, slide washing and scanning were performed following the manufacturers protocols (Agilent Technologies). Briefly, mRNA from 100 ng of total RNA was amplified, labeled with Cy3 and purified with columns. Labeled samples (600 ng) were hybridized on Agilent Human Gene Expression 8 × 60 K v3 microarrays. After 17 h, slides were washed and scanned using the Agilent Scanner version C (G2505C, Agilent Technologies). The fluorescence intensities of scanned images were extracted and pre-processed by Agilent Feature Extraction Software (v10.7.3.1).
Gene expression profiling was carried out using the one-color labelling method: labelling, hybridization, slide washing and scanning were performed following the manufacturers protocols (Agilent Technologies). Briefly, mRNA from 100 ng of total RNA was amplified, labeled with Cy3 and purified with columns. Labeled samples (600 ng) were hybridized on Agilent Human Gene Expression 8 × 60 K v3 microarrays. After 17 h, slides were washed and scanned using the Agilent Scanner version C (G2505C, Agilent Technologies). The fluorescence intensities of scanned images were extracted and pre-processed by Agilent Feature Extraction Software (v10.7.3.1).
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