Berlin 2018 – wissenschaftliches Programm

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BP: Fachverband Biologische Physik

BP 9: Postersession I

BP 9.41: Poster

Montag, 12. März 2018, 17:30–19:30, Poster A

Single-cell kinetics of siRNA-mediated mRNA degradation — •Rafał Krzysztoń1,2, Daniel Woschée1, Anita Reiser1,2, Gerlinde Schwake1, Helmut Strey3, and Joachim O. Rädler1,21Ludwig-Maximilians-Universität Munich (LMU), Geschwister-Scholl-Platz 1, Munich — 2Graduate School of Quantitative Biosciences (QBM), Geschwister-Scholl-Platz 1, Munich — 3Stony Brook University, Stony Brook, NY

RNA interference (RNAi) is a natural mechanism of post-transcriptional gene regulation and is underlying the therapeutic action of small interfering RNA (siRNA) directed against disease-related genes. Quantitative assessment of the siRNA knockdown efficiency is typically carried out at the population level. In contrast, direct measurement of the siRNA induced mRNA degradation requires time-resolved studies. Here we report on life cell imaging of the time-resolved expression and knockdown level after delivery of two mRNA reporter genes (eGFP, CayenneRFP) and delayed delivery of siRNA duplexes. Thousands of single cell time traces were recorded in parallel using micro-pattern assisted time-lapse microscopy (MPA-TLM) combined with automated image analysis. With the help of maximum likelihood fits to a mathematical translation model we yield scatter-plots of individual mRNA life-times and determine the siRNA meditated RISC activity as fold-change of mRNA degradation rate. Time-lapse imaging proves faster (<24hours) and more accurate (+/-1%) measurement of mRNA degradation and hence will allow new sensitive studies of sequence dependence RNAi

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DPG-Physik > DPG-Verhandlungen > 2018 > Berlin