Computational protocol: Development and Validation of an Haemophilus influenzae Supragenome Hybridization (SGH) Array for Transcriptomic Analyses

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Protocol publication

[…] Single-stranded cDNA was synthesized from the extracted RNA samples using the Roche Transcriptor First Strand Synthesis kit. Specific primers for the housekeeping and experimental genes were designed using Roche Probe Finder online software in order to design ∼75 bp amplicons. qRT-PCR was performed on the Roche Light Cycler 480 using a SYBR green master mix. Reactions were performed in a 20 µl volume containing 2 µl cDNA (1∶5 dilution) and primers at 0.5 µm each. Primer efficiency was determined by testing all primer pairs ahead of time with gDNA template. All reactions were measured in triplicate. The experimental data were normalized using the hpr and ldhA genes as internal standards. Independent data analysis was carried out using both the Pfaffl-ΔΔCT method with the Roche Light Cycler software as well as a linear regression method using the LinRegPCR software package. Fold changes presented are the mean results from both methods of analysis and from normalization against both of the housekeeping genes. [...] Images were processed to.pair files containing expression values for both sets of duplicate probes representing all the subclusters on the H. influenzae SGH array using the NimbleScan software. These.pair tables were merged with a reference list of subclusters that had been determined to be present in the CZ4126/02 genome (see above) in order to remove non-relevant probe/subcluster data. These parsed.pair files were then normalized within and across chips using a Robust Multichip Average (RMA) algorithm and quantile normalization using the NimbleScan v2.5 software followed by a median polish whereby the 3 to 13 probe values/subcluster were condensed to a single value (in duplicate) , . Duplicate probe-set values were treated as independent replicates. For comparison of technical and biological replicate data, CyberT was used to obtain Bayesian corrected p-values, Bonferroni corrected p-values and Benjamini-Hochberg values . Significance Analysis of Microarrays (SAM v3.0) was used to obtain lists of genes with associated permutation-based false discovery rates (FDR) . These data were combined and filtered in the following order: 1) SAM FDR <10%, Bayesian p-values<.05, Benjamini-Hochberg FDR<10%, Bonferroni corrected p-value<.05, raw values in at least one of the two conditions being compared >256 normalized intensity. […]

Pipeline specifications

Software tools LinRegPCR, SAM
Applications Gene expression microarray analysis, qPCR
Organisms Haemophilus influenzae