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
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