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Label-free detection of noncoding RNAs

Noij, Peter. Label-free detection of noncoding RNAs. 2012, PhD Thesis, University of Basel, Faculty of Science.

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Official URL: http://edoc.unibas.ch/diss/DissB_10046

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Abstract

Noncoding RNAs have emerged as highly conserved regulators for gene expression. Their detection is a biomarker for the identification and understanding of fundamental biological processes and diseases. They also play an important role in drug development.
To facilitate the detection of noncoding RNAs we set up a label-free direct binding assay. The assay is based on nanomechanical cantilever arrays for the detection of surface stress induced by immobilized biomolecules and their interaction partners. We used various means to significantly reduce the drift of the cantilever readout. Major improvements were achieved by tight control of temperature and mass transport which led to a faster system equilibration. Experimental protocols were improved to provide user-friendly and less time-consuming measurements. Further enhancements were achieved (i) by coating the entire cantilever array wafer with gold rather than individual cantilever arrays; (ii) by a directly implemented data analysis tool as real time feature of the measurement software. We succeeded to detect biomarker targets with high specificity in the picomolar range and we can easily distinguish perfect match from mismatches that hybridize with lower affinity.
We have demonstrated the detection of the biomarkers coagulation factor VII messengerRNA transcripts (F7) and microRNA-122 (miR-122) in proof of concept experiments with spike-in target in totalRNA background and cell lysates as well as the detection out of biologically relevant cell lysate samples with naturally occurring targets. Furthermore we have demonstrated the detection of miR-122 in acetaminophen treated rats’ plasma and therefore prove the cantilever array technology to be a useful tool for the label-free direct detection of noncoding RNAs.
Biacore’s surface plasmon resonance (SPR) system, the gold standard for label-free quantitative biomolecular interaction analysis, was used as reference parallel to the cantilever array technology (CLA). We compared the two fundamentally different systems, where the SPR measures the change in index of refraction in a boundary layer on the sensor surface and the CLA measures surface stress, in order to further support the interpretation of our results. The SPR can distinguish specific binding mostly by kinetic analysis and less by amplitude changes whereas the CLA offers to date less kinetic information but direct observation of amplitude changes. Plus, the CLA’s dynamic range and sensitivity is higher.
Advisors:Meyer, Ernst
Committee Members:Hegner, Martin
Faculties and Departments:05 Faculty of Science > Departement Physik > Physik > Nanomechanik (Meyer)
Item Type:Thesis
Thesis no:10046
Bibsysno:Link to catalogue
Number of Pages:76 S.
Language:English
Identification Number:
Last Modified:30 Jun 2016 10:51
Deposited On:18 Jan 2013 09:38

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