A Novel Nitrocellulose Filter Membrane Based Partitioning-Reverse Transcription Using Repora-6 RNA Aptamer In Detecting Erythropoietin (EPO)
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Date
2015-12
Authors
Ravinderan, Presela
Journal Title
Journal ISSN
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Publisher
Universiti Sains Malaysia
Abstract
Recombinant human EPO (rHuEPO), a mimetic of endogenous EPO is widely misused
by athletes as a performance enhancer, a phenomenon known as doping. Current
methods of detecting rHuEPO doping are via isoelectric-focusing (IEF)-doubleimmunoblotting,
Sarcosyl-PAGE (SAR PAGE) and lateral flow test, which are
antibody-based approach using anti-EPO antibody. The caveats associated with anti-
EPO antibodies have spurred the interest to adopt an alternative molecular recognition
element (MRE) in detecting EPO. Aptamers, as the nucleic acid-based MREs can
potentially alleviate the disadvantages inherent in anti-EPO antibodies. Previously,
REPORA-6 RNA aptamer was isolated against EPO with the dissociation constant of
25 nM. The objective of the study is to adopt REPORA-6 RNA aptamer in a
nitrocellulose filter membrane-based partitioning coupled with reverse transcription
assay in detecting EPO. REPORA-6 RNA aptamer was incubated with Epoetin-alpha
(EPO-α) before filtration through the 0.45 μm nitrocellulose membrane with the aid of
vacuum suction. EPO-bound RNA aptamers, retained on the surface of the membrane
was recovered using urea, ensued by ethanol precipitation. Reverse transcription was
carried out followed by PCR amplification till the band of the correct size appears (99
bp). Limit of detection (LOD) was defined as the lowest amount of EPO that results in
the PCR band with the intensity higher than the background non-specific binders of the
nitrocellulose membrane (0 nM of EPO). LOD was also approximated for the EPO
spiked into human serum and urine to mimic the actual clinical samples. LOD achieved
for EPO and for the EPO spiked into serum were 18.38 nM. For EPO spiked into urine,
LOD could not be determined due to the degradation of the RNA aptamer, which
suggests chemical modification for the enhanced stability. Despite this, nitrocellulose
filter membrane based partitioning-reverse transcription based on REPORA-6 RNA
aptamer could be a potential aptamer-based assay in the diagnostic detection of EPO.
Description
Keywords
A novel nitrocellulose filter membrane , based partitioning-reverse transcription