Figure 4.

Northern blot analysis of ATP7A-transcript levels. TRIZOL (Gibco) was used to isolate total RNA from lymphoblastoid cell lines from II-6, III-1, III-2, and III-3. mRNA was obtained using the PolyATtract mRNA Isolation System IV (Promega). A northern blot containing 2 μg of poly(A)⁺ from each family member was hybridized with a [³²P]-radiolabeled 1.2-kb cDNA probe, specific to exons 4–10 (nucleotides 1147–2335) of ATP7A. The primer sequences used to generate, via RT-PCR, the 1.2-kb product of normal lymphoblastoid cDNA were 5′- AATAGTGGCTGTATCACCGGG-3′ and 5′- GTACCAGCCTCCGAAAAACTG-3′. Subsequent hybridization with a [³²P]-radiolabeled β-actin probe (Clontech) demonstrated loading consistency of poly(A)⁺ RNA, in each lane (lower panel). Probes were radiolabeled by the rediprimeII Random Prime Labeling System (Amersham Pharmacia Biotech).