For microarray hybridization, Trizol extracted total RNA from mouse brain was further treated with DnaseI and purified by phenol/chloroform extraction and ethanol precipitation.
The total RNAs used for microarray hybridization were extracted from pools of zebra mussel feet with six individuals in each pool.
To ensure statistical significance, several separate microarray hybridizations and independently extracted mRNA or miRNA samples were used in all cases for the characterization of each genotype and/or experimental condition under study.
Microarray hybridization data were extracted from the Gene Expression Omnibus (GEO accession number GSE5350, file MAQC_AGL_123_60TXTs.zip, series "C": arrays AGL_3_C1.txt, AGL_3_C2.txt, AGL_3_C3.txt, AGL_3_C4.txt and AGL_3_C5.txt, series "D": AGL_3_D1.txt, AGL_3_D2.txt, AGL_3_D3.txt, AGL_3_D4.txt and AGL_3_D5.txt).
Finally, results obtained by qRT-PCR indicated that reliable microarray hybridizations can be achieved with rickettsial RNA extracted from multiple and independently obtained eschars.
Six independent microarray hybridizations were performed using total RNA preps extracted from pancreatic islets of WT and RasGrf1 KO mice.
Microarray hybridizations, image scanning and data extracting were performed by Nimblegen Inc. Background correction and data normalization were performed by Robust Microarray Analysis (RMA, [ 86]) in Bioconductor.
Single strain genomic DNA for microarray hybridization and PCR was extracted using E.Z.N.A Bacterial DNA kit (Omega Bio-Tek INorcrosscross, GA) with the following modifications: cell walls were digested with 7 μg/μL lysozyme and 0.5 U/μL mutanolysin for 20 min at 30°C, and DNA was eluted with ddH2O.
In order to avoid batch effects, we did not extract RNA until immediately prior to microarray hybridizations after all samples had been collected.
Sample preparation for microarray hybridization was performed by extracting, fragmenting and biotin-labelling 3 µg of genomic DNA from each strain according to Affymetrix standard protocols.
