Absolute quantitation of micro-RNAs using padlock probes and FRET (Niko Hildebrandt)


Friday 8th of December at 10.00
B7:113, Biomedical Centre (BMC), Uppsala

NanoBioPhotonics (nanofret.com), Institute for Integrative Biology of the Cell (I2BC), Université Paris-Saclay, Université Paris-Sud, CNRS, CEA, Orsay, France

(FRET) plays an important role for quantifying concentrations and distances in many fields of the life sciences. The application of time-resolved photoluminescence spectroscopy and microscopy for the analysis of FRET systems offers several advantages concerning versatility, sensitivity, and specificity. Luminescent lanthanide complexes, and in particular Tb complexes, exhibit extremely long luminescence lifetimes and multiple narrow emission bands over a broad spectral range. These photophysical features make them highly interesting FRET donors in combination with different FRET acceptors, such as organic dyes or semiconductor quantum dots

In this seminar, I will present different Tb-based FRET technologies for absolute quantitation of micro-RNAs. Tb-to-dye and Tb-to-quantum dot FRET was used to quantify various micro-RNAs with high sensitivity and high specificity. Both spectral (color) and temporal (time-gated detection) multiplexing were applied to detect different micro-RNAs from a single 150 µL sample using a clinical immunofluorescence plate reader (KRYPTOR – Thermo Fisher).

The best micro-RNA assays, with ultra-low detection limits (in the atto to femtomolar concentration range) for both micro-RNA and their DNA analogs, were accomplished by target-primed rolling-circle-amplification of padlock probes combined with Tb-to-dye FRET. Direct application in clinical diagnostics was demonstrated by quantitation of different micro-RNAs from RNA extracts of human plasma, tissue, and cell samples.

Event information flyer

 

Host: Caroline Gallant, SciLifeLab/UU, caroline.gallant@igp.uu.se