Quantification of human Aiolos splice variants by real-time PCR.
Aiolos is a transcriptional regulator of B cell development and belongs to the Ikaros family of chromatin remodelling transcription factors. All the members of Ikaros family produce multiple isoforms via alternative mRNA splicing. Altered expression of Ikaros isoforms has been found in patients with acute lymphoblastic leukemia but it is not studied whether the altered expression of Aiolos isoforms also has a role in the development of leukemias or lymphomas. We developed a quantitative real-time PCR application to detect the relative expression of Aiolos splice variants. The method is based on fluorescence resonance energy transfer (FRET)-labelled isoform specific hybridisation probes used with the LightCycler instrument. The isoform specificity is obtained by targeting the probes at the edges of chosen exons. The probes are here shown to represent a rapid, high throughput, specific and reproducible quantification method. We designed and optimised the analysis for a dominant negative Aiolos isoform, but the described method is applicable to any isoform-forming gene. This study shows that the real-time PCR with exon edge spanning probe pairs can be applied generally to reveal the importance of alternative splicing and the role of isoforms in normal development and diseases.
Veistinen
Elli
E
Department of Medical Microbiology, Turku Graduate School of Biomedical Sciences, University of Turku, Kiinamyllynkatu 13, Finland. elli.veistinen@utu.fi
Liippo
Jussi
J
Lassila
Olli
O
D017398:Alternative Splicing; D052160:Benzothiazoles; D003001:Cloning, Molecular; D015342:DNA Probes / Q000235:genetics; D003959:Diamines; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D051740:Ikaros Transcription Factor; D009693:Nucleic Acid Hybridization / Q000379:methods; D009930:Organic Chemicals; D020033:Protein Isoforms / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics; D011804:Quinolines; D012313:RNA / Q000737:chemistry / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015534:Trans-Activators / Q000032:analysis* / Q000096:biosynthesis / Q000235:genetics; D014157:Transcription Factors
D052160:Benzothiazoles; D015342:DNA Probes; D003959:Diamines; D005456:Fluorescent Dyes; C106285:IKZF3 protein, human; D009930:Organic Chemicals; D020033:Protein Isoforms; D011804:Quinolines; D015534:Trans-Activators; D014157:Transcription Factors; D051740:Ikaros Transcription Factor; C098022:SYBR Green I; D012313:RNA