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

For the best performance of in situ analysis of specific RNA expression in calcified tissues, it is necessary to choose an appropriate protocol to dec alcify the tissues. We evaluated the usefulness of various acid-based decal cifying reagents with reference to 28 S rRNA staining by in situ hybridizat ion using a thymine-thymine dimerized oligonucleotide probe. The reagents e valuated 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 retentio n of morphology and RNA were compared with EDTA-based solutions. When norma l mouse mandible was used as a model tissue, well-preserved morphology of a meloblasts 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 analys is 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 c orrelated with each other depending on decalcifying solutions.