ABSTRACT 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 lasermicrodissection (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 humanmaternal uterine scar tissue obtained fromuterotomy 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.
METHOD SUMMARY FFPE sample sections weremounted on glass slides and stained with hematoxylin and eosin (HE). A corresponding slide was stained according to Gomori trichrome for orientation. Regions of interest in HE-stained samples weremarked using lasermicrodissection (LMD) and subsequently scratched off the slide with a sterile scalpel. RNA extraction and cDNA synthesis were carried out, and quantitative real-time PCR with TaqMan probes was performed.
TWEETABLE ABSTRACT We describe a protocol for maximized RNA output from FFPE tissue delineated by laser microdissection devised to analyze gene expression of human maternal uterine scar tissue obtained from uterotomy scars of prior cesarean deliveries.
