|Application:||Gene expression microarray analysis|
|Number of samples:||6|
|Release date:||Sep 17 2010|
|Last update date:||Mar 22 2012|
|Diseases:||Infection, Malaria, Cerebral|
|Dataset link||Alterations in the Brain Transcriptome in Plasmodium Berghei ANKA Infected Mice|
We have used a previously published protocol (Iacobas et al., Physiol Genomics 2005) and a composite reference RNA sample (R) prepared in sufficient quantity for the entire experiment from ten adult mouse tissues (aorta, brain, heart, kidney, liver, lung, ovary/testicles, spleen, and stomach - equal amounts from males and females). This combination of source tissues provided a high diversity of genes expressed in the midrange of the detection system for the AECOM mouse cDNA microarrays. Briefly, 60μg total RNA, extracted in Trizol® (Invitrogen, Carlsbad, CA) from brains of three infected (I) and three control (C) mice, purified with RNeasy® mini kit (Qiagen, Valencia, CA), were reverse transcribed into cDNA incorporating fluorescent Cy3-dUTP. The composite reference was reverse transcribed to incorporate Cy5-dUTP. Each of the six Cy3-labeled brain extracts was co-hybridized overnight at 50°C against the Cy5-labeled reference with AECOM 32k Mouse oligonucleotide arrays, MO3 printing series (platform described in http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GPL5371). After hybridization, the slides were washed at room temperature, using solutions containing 0.1% sodium dodecyl sulfate (SDS) and 1% SSC (3M NaCl + 0.3M sodium citrate) to remove the non-hybridized cDNAs.
Dumitru Andrei Iacobas