Topo 2 plasmid

Mouse Trp53 complementary DNA was cloned from mouse brain total RNA by reverse transcription–polymerase chain reaction (Multiscribe; Applied Biosystems), as recommended by the manufacturer. Second-round amplification was performed with REDTaq (Sigma-Aldrich, Dorset, United Kingdom) and 0.5 μmol/L forward (5′-GGC-AAC-TAT-GGC-TTC-CAC-C-3′) and reverse (5′-CTC-CGT-CAT-GTG-CTG-TGA-C-3′) primers (based on accession number NM_001127233) using the following cycling parameters: 95°C for 5 minutes, 1 cycle; 95°C for 30 seconds; 65°C for 30 seconds; 72°C for 1 minute, 30 cycles; 72°C for 5 minutes. Each complementary DNA product was directionally cloned into TOPO II plasmid (Life Technologies, Carlsbad, CA), which incorporates T7 and SP6 RNA polymerase promoters flanking the cloning region defined by the HindIII or Xba 1 restriction enzyme site. Plasmids were sequenced, linearized, and transcribed with T7 (antisense) or SP6 (sense) RNA polymerases (Sigma) to yield digoxigenin-dUTP–labelled riboprobes in accordance with the manufacturer's protocol (Ambion, Life Technologies). Transcription was performed for 24 hours at 37°C. The template complementary DNA was digested away by RNase-free DNase (2 U/μL, 15 minutes), and the riboprobes were purified (Megaclear Purification and Filtration System; Ambion, UK) and quantified by spectrophotometry and electrophoresis.