Journal: BMC Genomics

Article Title: The human PINK1 locus is regulated in vivo by a non-coding natural antisense RNA during modulation of mitochondrial function

doi: 10.1186/1471-2164-8-74

Figure Lengend Snippet: 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.

Article Snippet: A pool of totalRNA from all the above cell lines was used for polyadenylated mRNA isolation using an oligo d(T) magnetic bead kit for mRNA isolation (Promega) Total RNA and mRNA was examined by Northern blotting.

Techniques: Northern Blot, Isolation, Quantitative RT-PCR, Nested PCR, Control, Sequencing, Agarose Gel Electrophoresis

Journal: BMC Genomics

Article Title: The human PINK1 locus is regulated in vivo by a non-coding natural antisense RNA during modulation of mitochondrial function

doi: 10.1186/1471-2164-8-74

Figure Lengend Snippet: Expression of PINK1, svPINK1 and naPINK1 in humans in vivo during gain in mitochondrial activity induced by 6 weeks of endurance training (a-d) . Total RNA was isolated from human muscle biopsies before and after activity to manipulate mitochondrial content. Expression of PINK1, svPINK1 and naPINK1 was achieved using quantitative real-time PCR (qRT-PCR). Mitochondrial Complex I activity demonstrates gain of mitochondrial content. Data are mean ± se and are presented as a percentage of the expression (or enzyme activity) before and after activity. (a) PINK1, svPINK1 and naPINK1 expression was determined after gain in mitochondrial activity (n = 24 for PINK1 and naPINK1, n = 23 for svPINK1 as the expression of svPINK1 in one paired sample was to low to measure). (b) Mitochondrial complex I activity and MTND4 mRNA were measured in muscle biopsies (n = 24) and the increase in enzyme activity is presented (c) Linear regression and correlation analysis of naPINK1 expression versus svPINK1 expression in human in vivo . Values compared were 18S rRNA adjusted CT-values from qRT-PCR analysis (n = 48). Dotted lines represent 95% confidence intervals. (d) Linear regression analysis of the changes in naPINK1 expression versus the changes in svPINK1 expression in the human in vivo model for mitochondrial biogenesis. Values compared were from the qRT-PCR analysis (18S adjusted CT-values before subtracted from 18S adjusted CT-values after 6 weeks of endurance training, n = 24).

Article Snippet: A pool of totalRNA from all the above cell lines was used for polyadenylated mRNA isolation using an oligo d(T) magnetic bead kit for mRNA isolation (Promega) Total RNA and mRNA was examined by Northern blotting.

Techniques: Expressing, In Vivo, Activity Assay, Isolation, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

Journal: BMC Genomics

Article Title: The human PINK1 locus is regulated in vivo by a non-coding natural antisense RNA during modulation of mitochondrial function

doi: 10.1186/1471-2164-8-74

Figure Lengend Snippet: Expression of naPINK1, PINK1 and svPINK1 following knockdown of naPINK1 with short interfering RNA (siRNA) (a-d) . Total RNA was isolated from neuroblastoma cell lines SK-N-MC and SH-SY5Y treated with two different siRNAs towards naPINK1 or an siRNA control which avoids targeting any known human sequence. Random hexamers were used in cDNA-synthesis when not otherwise stated and gene expression was determined using qRT-PCR. Data are presented as mean ± SE of the percentage mRNA abundance related to control siRNA treated samples in each experiment. (a) naPINK1 expression following AS-siRNA knockdown (n = 12 for control siRNA and AS siRNA 1, n = 11 for AS siRNA 2, one sample was excluded due to failed transfection). (b) PINK1 expression following AS-siRNA knockdown (n = 12 for control siRNA and n = 23 for pooled results from the two siRNAs). (c) svPINK1 following AS-siRNA knockdown (n = 12 for control siRNA and AS siRNA 1, n = 11 for AS siRNA 2). (d) To assess if naPINK1 and svPINK1 were polyadenylated and to exclude amplification of pre-mRNA species, naPINK1 and svPINK1 expression was determined in a subset of samples (n = 6) from which an additional cDNA synthesis was performed, this time using oligo d(T) 16 . Absolute values obtained for gene expression were similar to the random hexamer protocol while the extent of the interaction between naPINK1 and svPINK1 was arguably clearer.

Article Snippet: A pool of totalRNA from all the above cell lines was used for polyadenylated mRNA isolation using an oligo d(T) magnetic bead kit for mRNA isolation (Promega) Total RNA and mRNA was examined by Northern blotting.

Techniques: Expressing, Knockdown, Small Interfering RNA, Isolation, Control, Sequencing, cDNA Synthesis, Gene Expression, Quantitative RT-PCR, Transfection, Amplification, Random Hexamer