Assessment of decalcifying protocols for detection of specific RNA by non-radioactive in situ hybridization in calcified tissues

Abstract

For the best performance of in situ analysis of specific RNA expression in calcified tissues, it is necessary to choose an appropriate protocol to decalcify the tissues. We evaluated the usefulness of various acid-based decalcifying reagents with reference to 28 S rRNA staining by in situ hybridization using a thymine-thymine dimerized oligonucleotide probe. The reagents evaluated were 10% nitric acid, 10% HCl, 5% formic acid, 5% trichloroacetic acid, Morse's solution, Plank-Rychlo's solution, and K-CX solution, all of which are commonly used to decalcify tissues, and their effects on retention of morphology and RNA were compared with EDTA-based solutions. When normal mouse mandible was used as a model tissue, well-preserved morphology of ameloblasts was obtained from sections decalcified with Morse's solution, 10% HCl, Plank-Rychlo's solution, and K-CX solution, and best retention of 28 S rRNA was obtained with 5% formic acid and Morse's solution. We recommend Morse's solution to decalcify tissues to be processed for the rapid analysis of specific RNA expression. Indeed, we detected specific mRNAs strongly in sections treated with Morse's solution, and quantitative analysis showed that the ratio of signal intensities of 28 S rRNA and the specific mRNAs correlated with each other depending on decalcifying solutions.

The Expression of GM-CSF and Osteopontin in Immunocompetent Cells Precedes the Odontoblast Differentiation Following Allogenic Tooth Transplantation in Mice

“…Digoxigenin-labeled probe for OPN (Nomura et al 1993) was prepared according to the manufacturer's protocol. Following the fixation, the specimens were decalcified with Morse's solution (10% sodium citrate and 22.5% formic acid) for 24 hr (Shibata et al 2000), dehydrated through ethanol series and xylene, and embedded in paraffin. Then, 5-µm-thick paraffin sections were mounted on MAS-coated glass slides, deparaffinized, dehydrated, and predigested with proteinase K. The sections were then acetylated with 0.25% acetic anhydride in triethanolamine for 10 min and incubated overnight at 70C with hybridization buffer containing a digoxigeninlabeled probe for OPN.…”

Role of Bone Morphogenetic Proteins and Their Antagonists during Fracture Healing

“…In situ hybridization was performed as described previously with a slight modification 8) . After treatment with proteinase K (30 µg/ml) for 15 min at 37 , sections were hybridized with probes (1 µg per ml) for 16 h at 45 .…”

Gene Expression during Fracture Healing in Normal Mouse: In Situ Hybridization on Hard Tissues for Investigation of Regenerative Mechanisms.

“…We synthesized single-stranded DNA probes, more stable than RNA probes, by uni-directional PCR without in vitro transcription [21]. Furthermore, RNA of mouse bone tissue of the fracture model fixed with 4% PFA for 2 days was as well preserved after decalcification with 20% EDTA for 4 days as Morse's solution [32].…”