Detection of miRNA by SMART technology
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Sailis2017.pdf (4.268Mb)
Date
30/11/2017Item status
Restricted AccessEmbargo end date
31/12/2100Author
Sailis, Fiammetta
Metadata
Abstract
Aberrant expression of short non-coding micro RNAs (miRNA) in many human
diseases, along with remarkable stability in physiological media, has made them
attractive clinical biomarkers. In particular, miRNA-122 is substantially elevated
in plasma of patients with established drug-induced liver injury and can also be
used to identify early liver injury when current markers, such as alanine
aminotransferase (ALT), still show normal levels. The development of a rapid
test for miRNA-122 e.g. in drug poisoning would allow earlier and more sensitive
clinical diagnosis of liver injury.
Nucleic acids are traditionally analysed by polymerase chain reaction (PCR),
which has a high degree of sensitivity but suffers from high cost and is prone to
sample contamination.
The aim of this project is to develop a PCR free method to directly detect miRNA-
122 in biological samples using SMART technology.
The SMART technology takes advantage of dynamic chemistry for sequence
specific recognition of nucleic acids using aldehyde-modified nucleobases
(SMART nucleobases), and target-complementary peptide nucleic acid (PNA)
probe containing an “abasic” position (so called modified PNA probe).
In this study, this unique detection method was used in a fluorescent detection
with the use of light up probes, which are probes with an environmental dye as
nucleobase; a FRET system was also designed to allow the discrimination
between perfect match target and mismatched one.
The SMART technology was also transferred onto magnetic beads to develop an
ELISA like assay allowing sensitive and rapid detection of single stranded DNA
mimic of the miRNA-122. With its potential PCR free approach, this easily
adapted platform promises to transform and expand routine clinical diagnostic
testing and screening for circulating miRNAs.