Subject Area	Biochemistry, Genetics and Molecular Biology
More specific subject area	Pancreas, organoid culture, RNA biology
Protocol name	Method for Improved Integrity of RNA Isolated from Matrigel Cultures
Reagents/tools	Reagents: •MatrigelⓇ (Corning 354,230) •QIAzol Lysis Reagent (Qiagen 79,306) •Quant-iT™ RiboGreen™ RNA Reagent (Invitrogen™ R11491) • RNeasy Mini Kit (Qiagen 74,104) • Express SYBR™ GreenER™ qPCR Supermix (Invitrogen™ 1,178,401 K) • Dispase II (Sigma D4693)Equipment: •Agilent 2200 TapeStation system •Applied Biosystems, QuantStudio 7 Flex
Experimental design	After 4–6 days of culture, the culturing media is removed from each well and the cells in Matrigel are collected in two washing steps with cold PBS. After three centrifugation steps, the Matrigel is completely removed. The cell pellet is resuspended in lysis buffer and RNA isolation is performed.
Trial registration	If applicable, include clinical trial registry and number
Ethics	If applicable, include ethical details e.g. Patient informed consent, Ethics Review Board-competent authority approval, animal experimentation guidelines followed etc.
Value of the Protocol	•The method allows for the separation of Matrigel from cells that are cultured on the matrix •Isolation of RNA from cells removed from the matrix eliminates degraded RNA present in Matrigel, resulting in cellular RNA of high integrity