RNA preservation in decalcified cochlear samples.

HYPOTHESIS

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.

BACKGROUND

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.

METHODS

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.

RESULTS

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.

CONCLUSION

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.