Reverse transcription-polymerase chain reaction.

Material was isolated in RNAlater and quickly frozen. RNA extractions were performed using Trizol reagent (Invitrogen). Reverse transcriptase (RT) reactions were performed using Superscript III (Invitrogen) with random primers p(dN)₆ (Roche) and polymerase chain reaction (PCR) reactions using GoTaq Flexi polymerase (Promega). Primers located in exon 5 (5′-CTGGGCACCTGGACCTATGA-3′) and exon 7 (5′-GACGGTGATGATGATGGACG-3′) of Chrna1 (Scheffer et al. 2007) were used to attest of the presence of Chrna1 mRNA in the neuroepithelium of the apical region of the cochlea at different ages or in whole inner ear tissue. Additionally, primers located in exon 2 (5′-GTACAAGTCACCGTGGGTCTACAG-3′) and exon 5 (5′-CTCATCGAAGGGAAAGTGAGTGAC-3′) were used to confirm the lack of exon 4 in Chrna1^Δ4/Δ4 mice inner ear at E18.5 (223-bp amplicon instead of the 333-bp amplicon found in wild-type). This deletion was confirmed by sequencing of the amplification products. RT-PCR amplification of Hprt transcript with primers 5′-GCTGGTGAAAAGGACCTCT-3′ and 5′-CACAGGACTAGAACACCTGC-3′ (248-bp amplicon) was used as a positive control. Primers located in exons 3/4 (5′-GATGCAATGGTGCGACTATCGC-3′) and exon 6 (5′-GCCTCCGGGTCAATGAAGATCC-3′) of Chrng were used to confirm the expression of the γ-nAChRs subunit (360- and 204-bp amplicons, the latter resulting from alternative splicing of exon 5) (Mileo et al. 1995; Yamane et al. 2002). To avoid any contamination, the two muscles of the tympanic cavity, the stapedius muscle and the tensor tympani muscle, were carefully removed before the tissue was harvested.