NucleoSpin® RNA

Protocol tips

Upstream tips	Protocol tips	Downstream tips
- Aliquot rDNase and store at -20 °C.	- Dry the columns very well after the ethanol wash by adding an additional 2' >10000 rpm centrifuge with no buffer.

Upstream tips
- Aliquot rDNase and store at -20 °C.
Protocol tips
- Dry the columns very well after the ethanol wash by adding an additional 2' >10000 rpm centrifuge with no buffer.

Publication protocol

Rats of 21 and 70 days old exposed to 0 (control group), 0.1, 0.4, 1.0, and 1.5mg/kg MeHg were selected for microarray analysis of cerebellum and rats exposed to 0 and 1.5mg/kg MeHg for cerebrum. Prior to isolation of total RNA, cerebral and cerebellar brain regions were homogenized with a mortar and pestle under liquid nitrogen. Subsequently, the total RNAs derived from 3 to 6 biological replicate samples per treatment group (total 47 cerebellum and 21 cerebrum samples) were isolated using the NucleoSpin RNA II kit (Macherey-Nagel, Düren, Germany) RNA purification kit. RNA samples (eluted in water) were stored at −80ºC before further processing. The isolated RNA samples were sent to ServiceXS BV (Leiden, The Netherlands) where they were processed according to Affymetrix protocols. In brief, RNA concentration was determined by absorbency at 260nm with the Nanodrop ND-1000, and quality and integrity were verified using the RNA 6000 Nano assay on the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). Next, 2 μg of high-quality total RNA was used with the Affymetrix Eukaryotic One-Cycle Target Labeling and Control reagents to generate Biotin-labeled antisense cRNA. The quality of the cRNA was checked using the Agilent 2100 bioanalyzer. The labeled cRNA was further used for the hybridization to Affymetrix Rat230-2.0 Genome Genechips, harboring 31099 probe sets. After an automated process of washing and staining, absolute values of expression were calculated from the scanned array using the Affymetrix Command Console v1 software

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