The 5’end of the mouse Col11a2 gene was analyzed with primer extension assay, S1 nuclease protection assay, and RT-PCR. For the primer extension assay, 5 pmols of three different primers 5’ to the obtained cDNA sequence, based on the genomic sequences of the Rxrβ -Col11a2 intergenic region (see original article III for the location of the primers), were labeled for one hour at 37 °C with 50 µCi of [γ 32P]ATP using 15 units of T4 polynucleotide kinase (Gibco BRL) in a reaction volume of 10 µl. The labeled primers were mixed with 20 µg of mouse cartilage total RNA, isolated from 1 to 3 week old mouse xyphoid and/or rib cartilage with the Rapid Total RNA Isolation Kit (5 Prime-3 Prime, Inc.). The samples containing the labeled primers and total RNA were denatured at 65 °C for 30 minutes, and then allowed to cool slowly to room temperature for hybridization, after which they were precipitated and resuspended in 8 µl of water. The primer extension reaction was performed at 40 °C for one hour by adding 5 µl of the first-strand reaction mix (First-strand cDNA synthesis kit, Pharmacia), then terminated by adding 105 µl of a solution containing 100 mg/µl salmon sperm DNA and 20 µg/ml Rnase A (Pharmacia), incubating at 37 °C for 15 minutes, and precipitating the samples with ethanol. The primer extension products were analyzed on a 6% DNA sequencing gel, using dideoxynucleotide sequencing reactions of known DNA templates as size markers.