Date

2014

Author

Atılgan, Seren

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MicroRNAs are small non-coding RNAs that are involved in important regulatory pathways such as differentiation, development, metabolism, cell proliferation, and cell death. Several recent research show that deregulated expression of miRNAs has crucial roles in disease pathologies, mainly in cancer. Therefore, it is likely that the usage of miRNAs as diagnostic and prognostic biomarkers in patients and the development of various techniques for the detection of microRNA in clinical research will become widespread. In this study, we aimed to develop an alternative to existing tools for the detection of miRNAs using an array platform based on sandwich hybridization. The sandwich hybridization method is one of the techniques developed for faster, more sensitive and reliable detection. This method constitutes target miRNA to be detected and two oligonucleotide probes which are half complementary to target miRNA. In this method, the capture probe (P1) which is fixed to the glass surface with the help of cross-linkers, is specific to one part of the target molecule.And then, target molecule binds to the capture probe and the existence of target molecule is defined with the binding of signal probe (P2). Sandwich hybridization system allows direct use of RNA and does not require any labelling or cDNA synthesis steps which are necessary in the current sequence systems. In order to construct platform with sandwich hybridization, glass slides coated with poly-l-lysin were used. The sequences of probe 1 were attached to the surface by using different cross linkers such as Sulfo-EMCS and SM(PEG)n. After the determination of cross linker which fixed probe 1 to surface, several parameters were tried to determine blocking conditions, hybridization conditions like temperature, duration, oligonucleotide type, signal probe, probes’ concentration, washing conditions, and the sensitivity of platform and the optimization experiments were conducted. These experiments were conducted first for miR-21 and optimum hybridization signal was obtained with SM(PEG)2 as cross linker, 20 μM capture and 20 μM signal probe concentrations, and 0.1 μM platform sensitivity at 30 ⁰C. Then, these conditions were used for synthetic miRNA sequences and optimized for all miRNA probe sets. After these, the developed platform was tested whether it is unique for the targeted sequence or not by applying target sequence, mixed with several signal probes, to surface involving different capture probes. According to our results, optimum hybridization signal was obtained with 10 μM capture and 10 μM signal probe concentrations and 0.01 μM platform sensitivity at 45 ⁰C. The potential of platform workability with total RNAs obtained from different cell lines was tested. Finally, the comparison was made from the point of miRNA expression levels and signals between normal and cancer cells. Later, the obtained signals were compared with results taken from RT-PCR. The results indicated that the developed platform is specific enough to detect target miRNA sequences.

Subject Keywords

Nucleic acids., Nucleic acids, Non-coding RNA., Breast, Biochemical markers.

URI

http://etd.lib.metu.edu.tr/upload/12618056/index.pdf
https://hdl.handle.net/11511/24135

Collections

Graduate School of Natural and Applied Sciences, Thesis