Optimized RNA isolation of FFPE uterine scar tissues for RNA expression analyses delineated by laser microdissection
Alexander Paping,
Clara Basler,
Rebecca C Rancourt,
Loreen Ehrlich,
Kerstin Melchior,
Wolfgang Henrich,
Thorsten Braun
Affiliations
Alexander Paping
1Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin & Humboldt-Universität zu Berlin, Department of Obstetrics, Augustenburger Platz 1, Berlin, 13353, Germany
Clara Basler
2Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin & Humboldt-Universität zu Berlin, Division of ‘Experimental Obstetrics’, Augustenburger Platz 1, Berlin, 13353, Germany
Rebecca C Rancourt
2Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin & Humboldt-Universität zu Berlin, Division of ‘Experimental Obstetrics’, Augustenburger Platz 1, Berlin, 13353, Germany
Loreen Ehrlich
2Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin & Humboldt-Universität zu Berlin, Division of ‘Experimental Obstetrics’, Augustenburger Platz 1, Berlin, 13353, Germany
Kerstin Melchior
2Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin & Humboldt-Universität zu Berlin, Division of ‘Experimental Obstetrics’, Augustenburger Platz 1, Berlin, 13353, Germany
Wolfgang Henrich
1Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin & Humboldt-Universität zu Berlin, Department of Obstetrics, Augustenburger Platz 1, Berlin, 13353, Germany
Thorsten Braun
1Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin & Humboldt-Universität zu Berlin, Department of Obstetrics, Augustenburger Platz 1, Berlin, 13353, Germany
Samples for histological analyses are often formalin-fixed paraffin-embedded (FFPE) and slide-mounted, which complicates RNA extraction for many downstream molecular applications. Furthermore, when the region of interest is extremely small due to isolation with laser microdissection (LMD), extracting RNA of adequate quality and quantity is difficult. We describe an optimized protocol for maximizing RNA output from FFPE tissue devised to identify and analyze gene expression of human maternal uterine scar tissue obtained from uterotomy scars resulting from prior cesarean deliveries. Gomori trichrome staining allowed for region identification for LMD. Successful RNA isolation, reverse transcription and, importantly, quantitative real-time PCR (qRT-PCR) were performed. This report provides an optimized step-by-step protocol yielding sufficient RNA for qRT-PCR analyses from challenging tissue/LMD-FFPE samples.
