Decalcification of cochlear samples in Morse’s solution after methacarn fixation provides greater RNA quantification and morphologic preservation of cochlear structures as compared with EDTA and formic acid decalcifying solutions after methacarn fixation.
A variety of fixatives and decalcifying agents can fragment or chemically alter RNA in samples inhibiting their isolation and quantification. Morphologic alterations can also be observed in light microscopy analyses. The cochlea is embedded in the bone; hence, fixation and decalcification steps are mandatory to obtain histologic sections and preserve the cochlea for morphologic evaluation.
Cochlear samples obtained in a RNase-free environment were processed in 4 combinations of decalcifying agents in combination with methacarn fixation. Samples in Protocols 1, 2, and 3 were fixed in methacarn for 4 hours at 4°C, followed by decalcification at 4°C with Morse’s solution, 10% ethylenediaminetetraacetic acid, and 5% formic acid solution, respectively. Samples processed with protocol 4 were decalcified in Morse’s solution at 4°C followed by fixation for 4 hours at 4°C. Real-time PCR analysis was performed on total RNA extracted. Histology sections were evaluated for morphology preservation of cochlear structures.
RNA was isolated in all samples. Relative expression levels were greatest with Protocol 1 and lowest with Protocol 3. Morphology preservation was adequate with Protocols 1, 2, and 3.
Of the 4 protocols evaluated, methacarn fixation followed by decalcification in Morse’s solution provided the greatest genetic expression levels as well as the best tissue morphology preservation in the cochlea.
*Department of Surgical Research, McGill University; †Department of Surgery, Division of Urology, McGill University Health Centre and Research Institute; ‡Department of Physiology, and §Department of Otolaryngology–Head and Neck Surgery, McGill University, Montreal, Quebec, Canada
Address correspondence and reprint requests to: Sam J. Daniel, M.D., M.Sc., FRCSC., The Montreal Children’s Hospital, 2300 Rue Tupper, Rm. B-240, Montreal, QC, Canada, H3H 1P3; E-mail: firstname.lastname@example.org
This works is supported by an operating grant from the Canadian Institute of Health Research.
The authors disclose no conflicts of interest.