A Novel Nitrocellulose Filter Membrane Based Partitioning-Reverse Transcription Using Repora-6 RNA Aptamer In Detecting Erythropoietin (EPO)

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dc.contributor.authorRavinderan, Presela
dc.date.accessioned2019-07-16T07:51:02Z
dc.date.available2019-07-16T07:51:02Z
dc.date.issued2015-12
dc.description.abstractRecombinant 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.en_US
dc.identifier.urihttp://hdl.handle.net/123456789/8501
dc.language.isoenen_US
dc.publisherUniversiti Sains Malaysiaen_US
dc.subjectA novel nitrocellulose filter membraneen_US
dc.subjectbased partitioning-reverse transcriptionen_US
dc.titleA Novel Nitrocellulose Filter Membrane Based Partitioning-Reverse Transcription Using Repora-6 RNA Aptamer In Detecting Erythropoietin (EPO)en_US
dc.typeThesisen_US
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