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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 (3UTR)

regions of the target mRNA. miRNAs 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 non-

invasive 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. Real-

time 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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