Journal: Scientific Reports

Article Title: Exon-skipping and mRNA decay in human liver tissue: molecular consequences of pathogenic bile salt export pump mutations

doi: 10.1038/srep24827

Figure Lengend Snippet: For child 1, the ABCB11 splice-site mutation c.150 + 3A > C (red) is inherited by the mother whereas c.2783_2787dup5 (yellow) is transmitted by the father. ( a ) These mutations were detectable by sequencing of gDNA isolated from explanted liver of child 1. ( b , c ) For mRNA analysis, PCR forward primers ex2/4_for and ex3_for were used together with ex22/23_rev . Sequencing of BSEP exon 21 of both PCR products revealed the wildtype sequence. The GAGAT duplication (c.2783_2787dup5) was not detectable in mRNA transcripts with or without exon 3. ex: exon, for: forward primer, gDNA: genomic DNA, mRNA: messenger RNA, rev: reverse primer.

Article Snippet: Whole blood was used for genomic DNA (gDNA) extraction using MagNA Pure LC 2.0 DNA Isolation Kit I (Roche, Mannheim, Germany). gDNA from formalin-fixed paraffin-embedded (FFPE) liver tissue was isolated using EZ1 DNA Tissue Kit (Qiagen, Hilden, Germany) after pre-treatment of 10 μm slices with xylene and ethanol.

Techniques: Mutagenesis, Sequencing, Isolation

Journal: Scientific Reports

Article Title: Exon-skipping and mRNA decay in human liver tissue: molecular consequences of pathogenic bile salt export pump mutations

doi: 10.1038/srep24827

Figure Lengend Snippet: The splice-site mutation c.150 + 3A > C on the paternal allele is shown in red. The missense mutation c.2494C > T (p.R832C) inherited by the mother is displayed in purple. RNA from the liver biopsy was used for reverse transcription. ( a , b ) PCR forward primers ex2/4_for and ex3_for were combined with ex20/21_rev . Exon 20 was sequenced from both PCR products. Appearance of cytosine (C) or thymine (T) at position 2494 indicates the extent of exon-skipping caused by c.150 + 3A > C. ( a ) When primer ex2/4_for was used and hence exon 3 was absent, the main sequencing peak at c.2494 results from C. ( b ) In contrast, presence of exon 3 is associated with T to larger and C to lower amounts at that position. Occurrence of both alleles using ex3_for confirms that c.150 + 3A > C causes only partial exon-skipping in the patients’ liver tissues. for: forward primer, gDNA: genomic DNA, mRNA: messenger RNA, rev: reverse primer.

Article Snippet: Whole blood was used for genomic DNA (gDNA) extraction using MagNA Pure LC 2.0 DNA Isolation Kit I (Roche, Mannheim, Germany). gDNA from formalin-fixed paraffin-embedded (FFPE) liver tissue was isolated using EZ1 DNA Tissue Kit (Qiagen, Hilden, Germany) after pre-treatment of 10 μm slices with xylene and ethanol.

Techniques: Mutagenesis, Reverse Transcription, Sequencing