Figure 1

Mapping out the PINK1 locus (a). To validate the predicted transcripts from the PINK1 locus, Northern blotting and 5'-RLM RACE were performed (b-c). (a) A scaled overview of the PINK1 locus, presented along with the precise location of primer sites, siRNA target sites and Northern probe locations. The diagram represents the chromosomal coordinates for the PINK1 gene annotated in the Ensembl gene browser (20,832,535–20,850,591, chromosome 1). Arrows indicate direction of transcription (i.e PINK1 and svPINK1 are transcribed from left to right, while DDOST and naPINK1 are transcribed from right to left. (b) Total RNA was isolated from 4 neuroblastoma cell lines (SH-SY5Y (1), SK-N-SH (2), SK-N-AS (3), SK-N-F1 (4)) and poly adenylated mRNA was isolated using an oligo d(T) magnetic bead kit (from a pool of total RNA from all cell lines) and analyzed by Northern blotting with a double stranded probe targeting PINK1 exon 8, and thus would detect naPINK1 (4.4 kb), PINK1 (2.6 kb) and svPINK1 (1.6 kb). Three bands were detected of which two of them corresponded to the sizes of naPINK1 and PINK1, respectively. The third band is between 6 and 9 kb. It is plausible that it represents a larger transcript consisting of both the DDOST and the naPINK1 transcripts, which would result in a transcript of 6.4 kb. A fourth, band was detected in the mRNA preparation with a size that corresponds to svPINK1 (1.6 kb) consistent with the qRT-PCR abundance of svPINK1 (c) 5'-RLM RACE was performed to clone svPINK1 full-length cDNA. The svPINK1 RACE-product was confirmed using nested PCR. In parallel, as a size control, the same nested PCR was performed on a purchased clone (AK075225) containing the svPINK1 sequence. This clone was found using BLAST at the NCBI database. The size of the PCR products were determined by agarose gel analysis, which demonstrated bands at ~854 bp for both templates, consistent with the predicted size of the svPINK1 sequence, and the identify of both bands was then verified by sequencing.