Computational protocol: Selection of reliable reference genes for the normalisation of gene expression levels following time course LPS stimulation of murine bone marrow derived macrophages

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Protocol publication

[…] BMDMs were treated with vehicle or LPS, for 6 h and 24 h, and then RNA was isolated using Trizol reagent (Life Technologies) and the Qiagen Rneasy Plus Mini Kit (Qiagen). The aqueous phase of the Trizol preparation was obtained, according to the manufacturer’s instructions, and was applied to the gDNA-eliminating column of the RNeasy Plus Mini Kit, and then RNA was isolated according to the instructions of the manufacturer (Qiagen). RNA was submitted to the Ramaciotti Centre for Genomics (UNSW, Australia) for gene expression microarray analyses using the Affymetrix Mouse Gene 2.1ST array. Array data were analysed using Partek Genomics Suite (Partek Inc. USA). Data files were grouped according to treatment (control or LPS) and individual gene lists for 6h and 24h LPS stimulation were generated by one way ANOVA comparison of the two groups, with a fold change cut off of −1.NormFinder [] and BestKeeper [] were used according to the instructions supplied. This required that raw Ct values were transformed to different input formats for GeNorm and NormFinder analyses. For analysis using BestKeeper software, raw Ct values were inputted. For the statistical comparison of two groups, an unpaired, two-tailed t test was used in GraphPad Prism 7 software (GraphPad). P values indicated in the figure legends. […]

Pipeline specifications

Software tools Partek Genomics Suite, NormFinder, BestKeeper
Applications Gene expression microarray analysis, qPCR
Organisms Mus musculus, Homo sapiens