7 protocols
normalization data transformation protocol
The moderated t-test included in the VM2 package was finally applied on each gene and raw p-values were adjusted for multiple testing by the Benjamini and Yekutieli (BY) method, which controls the false discovery rate (FDR). We considered genes as being differentially expressed when the BY p value less than or equal to = 0.05. Only genes with an expression fold change (FC) greater than or equal to 2 were kept in the present analysis.
normalization data transformation protocol
Statistical analyses were carried out with the R software (http://www.R-project.org) and Bioconductor packages (http://www.bioconductor.org). Normalization was first performed on all spots using the normalizeWithinArrays function of the VM2 package. The intensity mean to all the probes corresponding to each gene was obtained. After, the final result was reported as the intensity mean of all hybridizations (hybridization A to F).
array scanning and feature extraction protocol
After washing and drying, slides were scanned (GenePix 4200 A scanner, Axon) and images were analysed (GenePix6 software, Axon). The Eh1blok_Complet_Nov10.gal file was used as a grid templated. The photomultiplier (PMT) gains were set for all slides. For each spot, the fluorescence value corresponded to the median pixel intensity within the spot (the GenePix F635 and F532 median columns). Empty and flagged spots were excluded.
hybridization protocol
Labelled cDNA samples were hybridized according to the manufacturers protocol (Two-color microarray-based gene expression analysis protocol, Agilent).
nucleic acid labeling protocol
The RNA was reverse-transcribed using Superscript III Reverse Transcriptase (45-0039 Invitrogen), according to the manufacturers protocol. cDNA was then labelled using SuperScript indirect cDNA labeling System (Invitrogen).
treatment protocol
E. histolytica (1.6X105) grown in axenic culture and treated or not with metronidazole were lysed with Trizol reagent (Invitrogen), and total RNA isolated according to the manufacturers protocol.
nucleic acid extraction protocol
RNA was purified from approximately 4 million of trophozoites by using 10 mL of Trizol reagent (Invitrogen) according to the manufacturers protocol. The quality and integrity of purified RNAs was checked by spectrophotometry at 230, 260, 280 and 320 nm, electrophoresis on a 0.8% agarose gel and an assay on a Bioanalyser 2100 (Agilent).