Computational protocol: Probiotic Lactobacillus fermentum strain JDFM216 stimulates the longevity and immune response of Caenorhabditis elegans through a nuclear hormone receptor

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Protocol publication

[…] Synchronized populations of fer-15;fem-1 mutants were harvested to examine gene expression in the host in response to preconditioning with JDFM216 or OP50 for 24 h. Total RNA from worms was immediately isolated using TRIZOL reagent (Invitrogen), according to the manufacturer’s instructions, and purified using an RNeasy Mini Kit (Qiagen) including an on-column DNase digestion with RNase-free DNase (Qiagen) coupled with a mini bead beater (Biospec, Bartlesville, OK). The synthesis and fragmentation of target cRNA probes and hybridization were performed using Agilent’s Low RNA Input Linear Amplification kit (Agilent Technology, Santa Clara, CA), according to the manufacturer’s instructions. The fragmented cRNA was resuspended in 2×hybridization buffer and then applied with a pipette directly onto C. elegans 4 × 44 K microarray chip (Agilent Technology). The arrays were hybridized at 65 °C for 17 h using Agilent’s Hybridization Oven. The hybridized microarrays were washed according to the manufacturer’s protocol (Agilent Technology) and were analyzed using GenePix Pro 6.0 (Axon Instruments, Foster City, CA). The average fluorescence intensity for each spot was calculated, and local background was subtracted. All data normalization and selection of fold-changes were performed using GeneSpring 7.3.1 (Agilent Technology). Intensity-dependent normalization [locally weighted regression scatter plot smoothing (LOWESS)] was performed, wherein the ratio was reduced to the residual of the LOWESS fit of the intensity versus ratio curve. Averages of the normalized ratios were calculated by dividing the average normalized signal channel intensity by the average normalized control channel intensity. A gene was considered differentially expressed when the p-value for comparing two chips was <0.05 (to ensure that the change in gene expression was statistically significant and that false positives were <5%). Functional annotations of genes from the Gene Ontology Consortium were retrieved with GeneSpring GX 7.3. Genes were based on searches of DAVID (http://david.abcc.ncifcrf.gov/) and Medline (http://www.ncbi.nlm.nih.gov/) databases. […]

Pipeline specifications

Software tools GenePix Pro, GeneSpring GX, DAVID
Application ChIP-on-chip analysis