Direct quantification of circulating MiRNAs in different stages of nasopharyngeal cancerous serum samples in single molecule level with total internal reflection fluorescence microscopy

See Lok Ho, Ho Man Chan, Amber Wai Yan Ha, Ricky N S WONG*, Hung Wing LI

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

27 Citations (Scopus)

Abstract

MicroRNAs (miRNAs) are small noncoding RNAs that regulate human gene expression at the post-transcriptional level. Growing evidence indicates that the expression profile of miRNAs is highly correlated with the occurrence of human diseases including cancers. Playing important roles in complex gene regulation processes, the aberrant expression pattern of various miRNAs is implicated in different types and even stages of cancer. Besides localizing in cells, many of these miRNAs are found circulating around the body in a wide variety of fluids such as urine, serum and saliva. Surprisingly, these extracellular circulating miRNAs are highly stable and resistant to degradation, and therefore, are considered as promising biomarkers for early cancer diagnostic via noninvasive extraction from body fluids. Unfortunately, the abundance of these small RNAs is ultralow in the body fluids, making it challenging to quantify them in complex sample matrixes. Establishing a sensitive, specific yet simple assay for an accurate quantification of circulating miRNAs is therefore desirable. Our group previously reported a sensitive and specific detection assay of miRNAs in single molecule level with the aid of total internal reflection fluorescence microscopy. In this work, we advanced the assay to differentiate the expression of a nasopharyngeal carcinoma (NPC) up-regulator hsa-mir-205 (mir-205) in serum collected from patients of different stages of NPC. To overcome the background matrix interference in serum, a locked nucleic acid-modified molecular beacon (LNA/MB) was applied as the detection probe to hybridize, capture and detect target mir-205 in serum matrix with enhanced sensitivity and specificity. A detection limit of 500 fM was achieved. The as-developed method was capable of differentiating NPC stages by the level of mir-205 quantified in serum with only 10 L of serum and the whole assay can be completed in 1 h. The experimental results agreed well with those previously reported whereas the quantity of miR-205 determined by our assay was found comparable to that of quantitative reverse transcription polymerase chain reaction (qRT-PCR), supporting that this assay can be served as a promising noninvasive detection tool for early NPC diagnosis, monitoring and staging.

Original languageEnglish
Pages (from-to)9880-9886
Number of pages7
JournalAnalytical Chemistry
Volume86
Issue number19
DOIs
Publication statusPublished - 7 Oct 2014

Scopus Subject Areas

  • Analytical Chemistry

Fingerprint

Dive into the research topics of 'Direct quantification of circulating MiRNAs in different stages of nasopharyngeal cancerous serum samples in single molecule level with total internal reflection fluorescence microscopy'. Together they form a unique fingerprint.

Cite this