Planktonic and biofilm cells were grown as described above in flasks (for planktonic) or in bovine-serum coated 6 well plates (for biofilms). Biofilm cells were harvested with a sterile transfer pipet, biofilm or planktonic cells were centrifuged at 3,000 × g for 5 min, supernatant was removed and cell pellets were frozen in liquid nitrogen and stored at −80°C. Total RNA was extracted using the Ambion RiboPure-Yeast RNA kit (AM1926) according to the manufacturer’s protocol. Synthesis of cDNA and dye coupling was performed as described in (Nobile et al., 2009 (link)). Gene expression microarrays from Agilent Technologies (AMID #020166), were used as described in (Nobile et al., 2012 (link)). LOWESS normalization of microarray data was performed as previously described (Lohse and Johnson, 2010 (link)). At least two independent replicates were performed at each timepoint. Microarray data is reported in Dataset 1 and raw gene expression array data is stored at the Gene Expression Omnibus (www.ncbi.nlm.nih.gov/geo , accession #GSE61143).
Gene expression microarrays
Manufactured by Agilent Technologies
Gene expression microarrays are a type of lab equipment used to measure the expression levels of genes in a sample. They consist of a collection of microscopic DNA spots attached to a solid surface, each representing a specific gene. By analyzing the pattern of gene expression, researchers can gain insights into cellular processes and identify changes associated with various biological conditions.
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Lab products found in correlation
4 protocols using gene expression microarrays
1
Transcriptome Analysis of Planktonic and Biofilm Cells
2
Mouse Tissue Total RNA Extraction and Microarray
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Total RNA from mouse tissues was extracted in the morning (ZT = 1–4) with the miRNeasy Mini kit (QIAGEN), labelled with the Lowinput Quick Amp labelling Kit (Cy3 mono color, Agilent) and hybridized to gene-expression microarrays (Agilent). Fluorescent signals were quantified with an Agilent microarray laser scanner. The microarray data and protocols were deposited in the Arrayexpress database (accession No. E-MTAB-3820). We performed gene enrichment analysis on METACORE-annotated process networks (http://thomsonreuters.com/metacore ), using the hypergeometric test after correction for multiple testing and calculation of the false discovery rate (FDR). Differential metabolomics were performed as detailed in Supplementary Methods .
3
RNA Extraction and Microarray Analysis of Planktonic and Biofilm Cells
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Planktonic and biofilm cells were grown as described above in flasks (for planktonic) or in bovine‐serum coated 6 well plates (for biofilms). Biofilm cells were harvested with a sterile transfer pipet, biofilm or planktonic cells were centrifuged at 3,000× g for 5 min, supernatant was removed and cell pellets were frozen in liquid nitrogen and stored at −80°C. Total RNA was extracted using the Ambion RiboPure‐Yeast RNA kit (AM1926) (Life Technologies, Grand Island, New York, USA) according to the manufacturer's protocol. Synthesis of cDNA and dye coupling was performed as described in Nobile et al. (2009 ). Gene expression microarrays from Agilent Technologies (AMID #020166) were used as described in Nobile et al. (2012 ). LOWESS normalization of microarray data was performed as previously described (Lohse and Johnson, 2010 ). At least two independent replicates were performed at each time point. Microarray data are reported in Dataset 1, and raw gene expression array data are stored at the Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo , accession #GSE61143).
4
Tobacco Leaf Gene Expression Profiling
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Gene expression profiles of tobacco leaves were assessed via Agilent Tobacco Gene Expression Microarrays (G2519F). cRNA labeling, hybridization and microarray processing were carried out at CapitalBio Corporation (Beijing, China) and labeling reactions were carried out using a CapitalBio cRNA Amplification and Labeling Kit (CapitalBio Corp.) in the presence of a fluorescent dye (Cy3-dCTP). All samples were labeled with Cy3 in this study. In order to rule out bias attributed to the dye, the sample labeling with these dyes was reversed (Yu et al., 2010 (link)). Hybridization was performed at 42°C for 16 h in a CapitalBio BioMixer II Hybridization Station (CapitalBio Corp.). Microarray slides were scanned with a LuxScan 10KA confocal laser scanner (CapitalBio Crop.) at 535 nm for Cy3 and 625 nm for Cy5 after washing. The obtained images were then evaluated with LuxScan 3.0 software (CapitalBio Corp.) which employs the LOWESS method (locally weighted scatter plot smoothing regression) (Yang et al., 2002 (link)) to minimize differences of dye incorporation efficiency in a two-channel microarray platform. Spots with fluorescence signal intensities of < 800 U (after subtracting the background in both channels) were regarded as empty spots and not analyzed further.
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