Rna Labeling And Hybridization

cDNA was generated by in vitro transcription from 35 |g total RNA. RNA was combined with 1 |g oligo dT12-18 primer (Amersham Pharmacis Biotech, Piscataway, NJ) and 10 U RNase-inhibitor (Invitrogen, Carlsbad, CA) and heated to 70°C for 10 min. Samples were chilled to 4°C for 2 min, then first strand buffer (50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2; Invitrogen), 11 mM dithiothreitol (Invitrogen), 2.2 nM FluoroLink Cy3-deoxy (d) UTP or Cy5-dUTP (Amersham Pharmacia Biotech), dNTP mix (0.7 mM dATP, 0.7 mM dGTP, 0.7 mM dCTP, 0.4 mM dTTP; Amersham Pharmacia Biotech), and 2 |l SUPERSCRIPT™ II Reverse Transcriptase (Invitrogen) were added. After incubation at 42°C for 1.5 h, another 2-|l aliquot of SUPERSCRIPT™ II Reverse Transcriptase was added, and samples were incubated for an additional 1.5 h at 42°C. After cDNA synthesis, RNA was degraded by addition of 30 |l of 0.1 M NaOH and incubation for 30 min at 70°C. The pH was neutralized by addition of 30 |l of 0.1 M HCl, and Cy3- and Cy5-labeled samples were pooled. Microcon-30 filters (Millipore Corp., Bedford, MA) were used to remove unincorporated label. Ten |g human COT1 DNA (Invitrogen) per 10 |g RNA and 20 ig yeast tRNA (Invitrogen) were added to the probe to limit nonspecific binding. Hybridization solution (3 x SSC, 2 x Denhardt's, and 0.8% SDS) was added to the samples and they were boiled for 2 min, and then purified with a 0.45-|m filter (Millipore Corp., Bedford, MA).

Roughly 7000 rat clone cDNAs (Research Genetics, Huntsville, AL) (http://dir.niehs.nih.gov/microarray/chips.htm) were printed on glass slides as described in Hamadeh et al. (14). The methods used to produce the chips are available at

http://dir.niehs.nih.gov/microarray/methods.htm. The cDNA clones were sequence verified and annotated according to UniGene (15).

The labeled cDNA, representative of cellular mRNA, was applied to cDNA microarray chips, covered by a cover-slide, and incubated for 24 h in a humidified chamber at 65°C. At the end of the incubation period, slides were inverted in 0.5 x SSC, 0.01% SDS for 5 min to remove cover-slides. After that, slides were washed in 0.5 x SSC, 0.01% SDS for 5 min, and in 0.06 x SSC for 5 min. After washing, slides were dried by spinning for 3 min at 1000 g.

Each individual treated RNA sample was hybridized against its time-and dose-matched control pool in duplicate with reversal of the fluorescent Cy3 or Cy5 dyes. This resulted in a total of 6 microarray chips per dose and time period.

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