Merging Absolute and Relative Quantitative PCR Data to Quantify STAT3 Splice Variant Transcripts. - Université de Lille Accéder directement au contenu
Article Dans Une Revue Journal of visualized experiments : JoVE Année : 2016

Merging Absolute and Relative Quantitative PCR Data to Quantify STAT3 Splice Variant Transcripts.

Résumé

Human signal transducer and activator of transcription 3 (STAT3) is one of many genes containing a tandem splicing site. Alternative donor splice sites 3 nucleotides apart result in either the inclusion (S) or exclusion (ΔS) of a single residue, Serine-701. Further downstream, splicing at a pair of alternative acceptor splice sites result in transcripts encoding either the 55 terminal residues of the transactivation domain (α) or a truncated transactivation domain with 7 unique residues (β). As outlined in this manuscript, measuring the proportions of STAT3's four spliced transcripts (Sα, Sβ, ΔSα and ΔSβ) was possible using absolute qPCR (quantitative polymerase chain reaction). The protocol therefore distinguishes and measures highly similar splice variants. Absolute qPCR makes use of calibrator plasmids and thus specificity of detection is not compromised for the sake of efficiency. The protocol necessitates primer validation and optimization of cycling parameters. A combination of absolute qPCR and efficiency-dependent relative qPCR of total STAT3 transcripts allowed a description of the fluctuations of STAT3 splice variants' levels in eosinophils treated with cytokines. The protocol also provided evidence of a co-splicing interdependence between the two STAT3 splicing events. The strategy based on a combination of the two qPCR techniques should be readily adaptable to investigation of co-splicing at other tandem splicing sites.
Fichier principal
Vignette du fichier
Merging-absolute-relative-quantitative-pcr-data-to-quantify-stat3 2016.pdf (1.6 Mo) Télécharger le fichier
Origine Fichiers éditeurs autorisés sur une archive ouverte

Dates et versions

hal-04507254 , version 1 (16-03-2024)

Licence

Identifiants

Citer

Keren B Turton, Stéphane Esnault, Larissa P Delain, Deane F Mosher. Merging Absolute and Relative Quantitative PCR Data to Quantify STAT3 Splice Variant Transcripts.. Journal of visualized experiments : JoVE, 2016, Journal of visualized experiments : JoVE, ⟨10.3791/54473⟩. ⟨hal-04507254⟩

Collections

UNIV-LILLE
2 Consultations
5 Téléchargements

Altmetric

Partager

Gmail Mastodon Facebook X LinkedIn More