Quantifying TRAP mRNA+ Osteocytes in Bone

TRAP gene expression was studied by in situ hybridization. A gene sequence for rat TRAP [28 (link)] was amplified by conventional PCR using cDNA from rat bone and oligonucleotide forward and reverse primers: rnTRAP.for 5′-ACGCCAATGACAAGAGGTTC-3′, rnTRAP.rev 5′-ACATAGCCCACACCGTTCTC-3′ (Life Technologies, Carlsbad, CA, USA) and cloned in a Dual Promoter TA Cloning Kit (Life Technologies). The cloned insert was sequenced to establish the orientation (Seqlab, Göttingen, Germany). A digoxigenin (DIG)–conjugated complementary RNA probe was synthesized using T7 or Sp6 polymerase to yield the probe in the sense or antisense direction (DIG-labeling kit; Roche Diagnostics, Oslo, Norway). Longitudinal sections from the tibial diaphysis (Ovx-D/sham) and femoral diaphysis (experimental rickets) were subjected to hybridization following our established protocol [29 (link)]. TRAP mRNA⁺ osteocytes were quantified in cortical bone within 4–10 mm from the proximal EMB by point counting in a squared grid. Three sections were examined from each animal and their means compared between the groups. Tibial diaphyses were examined twice with an interclass correlation of p < 0.001 and Cronbach’s alfa of 0.94. Staining of osteoclasts from the femoral metaphysis in healing for 72 h was used as a positive control. The sense probe did not show any staining.