Protocol 43 RNA labeling and hybridization

Ten |g total RNA were mixed with 1 |g (dT)17 primer, incubated at 70°C for 10 min and cooled on ice. The labeling reaction was performed in 12.5 |l containing 2.5 |l 5 x RT buffer (Invitrogen, Karlsruhe, Germany), 1.25 |l 0.1 M DTT, 1 |l dNTP mix (5 mM each dATP, dGTP, dTTP), 0.5 |l 3 mM dCTP, 0.5 |l (20 U) RNasin, 0.5 |l 1 mM Cy3- or Cy5-labeled dCTP (Amersham) and 1 |l (100 U) Superscript II reverse transcriptase (Invitrogen). The mixture was incubated for 1 h at 42°C, and the reaction was stopped by addition of 1.25 |l 50 mM EDTA (pH 8). The RNA was removed by hydrolysis with 5 |l 1 M NaOH at 65°C for 10 min, followed by neutralization with 1 |l 5 M acetic acid. Cy3- and Cy5-labeled samples were combined, precipitated with 100 |l isopropanol at -20°C for 30 min and centrifuged at 13,000 g for 15 min. The pellets were washed with 70% ethanol, air dried, and dissolved in 30 |l 1 x DIG-Easy hybridization buffer (Roche Diagnostics, Mannheim, Germany), containing 5 x Denhardt's solution and 10 ng |l-1 Cot1-DNA (Invitrogen). The sample was heat denatured (65°C, 2 min) and hybridized to the DNA on microarrays in a hybridization chamber (Corning, Acton, MA) overnight at 37°C. Unspecific probe binding was removed by washing with 1 x SSC/0.1% SDS (15 min) and 0.1 x SSC/0.1% SDS (10 min) followed by cleaning with 70% ethanol, 95% ethanol, and isopropanol before drying with pressurized air.

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