Real-time quantitative RT-PCR for low-abundance transcripts in the inner ear: analysis of neurotrophic factor expression

Abstract

Real-time quantitative reverse transcription-PCR is a highly sensitive technology that allows high throughput quantification of gene expression. Application of this technique to the inner ear is potentially very important, but is not straightforward because tissue harvesting can be challenging, RNA yield from individual inner ears is low, and cDNA synthesis from scant RNA can be inefficient. To overcome these challenges, we tested many parameters and reagents, and developed an approach to reliably quantitate small changes in low-abundance transcripts. Using this technique we demonstrate the presence and quantify amounts of the neurotrophic factors neurotrophin 3 (NT-3), brain-derived neurotrophic factor (BDNF) and glial cell-line-derived neurotrophic factor (GDNF), in the cochlea and vestibular end organs of postnatal murine inner ear (P26). We show that out of the factors tested, BDNF is the only one differentially expressed between the cochlea and vestibular end organs, being 23.4±0.3 times more abundant in the vestibular end organs. Within the cochlea, GDNF gene expression is 4.9±0.2 times greater than NT-3 expression. Within the combined vestibular end organs, BDNF expression is 43.0±1.5 times greater than NT-3 expression. Our results suggest that neurotrophic factors continue to play a role in the postnatal inner ear, in addition to their previously shown essential role during development.