Assay of micro RNA expression in Urine

Micro RNAs (miRNAs) are short non-coding RNA oligonucleotides that negatively regulate gene expression by incomplete base pairing with the untranslated (3’UTR) regions of the target mRNA. miRNA’s implicates to a variety of basic cellular functions like cell development, differentiation, metabolism, apoptosis and proliferation (Huang, Y et al, 2009). They are potential biomarkers in several human diseases and cancers. (Bartels, C.L & Tsongalis, G.J, 2009). Several miRNAs have intriguing specific expression as they have been identified in many different types of cancers including pancreatic cancer, renal cancer, breast and colon cancer to determine the blood based biomarkers. Alterations in the levels of miRNAs have been observed in several types of cancer and high copy number variation appears to be a common cause and over expression of certain miRNAs leads to oncogenesis. (Wijnhoven, et al., 2007).

The urine derived miRNAs holds a gateway to explore the possible outcomes for early prognosis for many potential diseases. Urine based biomarkers can be evaluated as noninvasive indicators of kidney cancers. Exosomes are tiny vesicles are present abundantly in urine and can be acquired my non-invasive methods and are enriched with miRNAs and assists in robust assays that would be essential as predictive biomarkers of renal cancer. (Pisitkun T, et al., 2006)

The objective of this project is to optimize the best method of purification of exosomes also, to examine if the yield of exosomes can be improved for the detection of miRNAs

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by performing different assays, also to effectively quantify the amount of exosomes by each method. Most miRNAs are hallmark of several diseases, including cancer. Realtime PCR was used to quantify the gene expression levels and the quality of RNA achieved was detected by Agilent Bioanalyser. The purity of exosomes can also be validated by Scanning Electron Microscopy and Transmission Electron Microscopy. Building on the different methods of isolation, the novel approach to be able to characterise the most effective method of isolation renders an avenue for investigating the roles of proteins present within these vesicles. This study also involved the involvement of Tamm Horsfall protein; which significantly reduces the yield of exosomes on isolation. The possible elimination of this protein by the addition of Dithiothreitol (DTT) is also studied. The direct involvement of miRNAs in causing the disease can be used for identification of the certain biomarkers for the disease and in future, the results of this study can be used as diagnostic markers for cancer.

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