Abstract
This paper describes a straightforward electrochemical method for rapid and robust urinary microRNA (miRNA) quantification using disposable biosensors that can discriminate between urine from diabetic kidney disease (DKD) patients and control subjects. Aberrant miRNA expression has been observed in several major human disorders, and we have identified a urinary miRNA signature for DKD. MiRNAs therefore have considerable promise as disease biomarkers, and techniques to quantify these transcripts from clinical samples have significant clinical and commercial potential. Current RT-qPCR-based methods require technical expertise, and more straightforward methods such as electrochemical detection offer attractive alternatives. We describe a method to detect urinary miRNAs using diazo sulfonamide-modified screen printed carbon electrode-based biosensors that is amenable to parallel analysis. These sensors showed a linear response to buffered miR-21, with a 17 fM limit of detection, and successfully discriminated between urine samples (n = 6) from DKD patients and unaffected control subjects (n = 6) by differential miR-192 detection. Our technique for quantitative miRNA detection in liquid biopsies has potential for development as a platform for non-invasive high-throughput screening and/or to complement existing diagnostic procedures in disorders such as DKD.
Highlights
MicroRNAs are short single-stranded noncoding RNAs that regulate the expression of most mammalian protein coding genes
This paper describes a straightforward electrochemical method for rapid and robust urinary microRNA quantification using disposable biosensors that can discriminate between urine from diabetic kidney disease (DKD) patients and control subjects
The following materials were obtained from the corresponding suppliers: DNA oligonucleotides, K3[Fe(CN)6], K4[Fe(CN)6], streptavidin peroxidase polymer (1 mg mLÀ1), chloroform, diethyl ether, ethanol, molecular biology grade water, from Merck (Watford, UK); tetramethylbenzidine (TMB) substrate solution, KCl, PCl5, NaNO2 and RT-qPCR reagents from Thermo Fisher Scienti c (Gloucester, UK); RNA oligonucleotides from Integrated DNA Technologies (Leuven, Belgium); 4-amino-3-hydroxy-1-napthalene sulfonic acid (ANSA) from Fluorochem (Glossop, UK) and miRNeasy mini kit for miRNA extraction from Qiagen (Manchester, UK)
Summary
MicroRNAs (miRNAs) are short single-stranded noncoding RNAs that regulate the expression of most mammalian protein coding genes. Aberrant miRNA expression pro les have been observed in major disorders including cancer, cardiovascular disease, atherosclerosis, diabetes and chronic kidney disease that requires treatment by dialysis or transplantation.[1,2,3,4]. We showed that miR-21 may predict early renal transplantation outcomes.[7] In peritoneal dialysis effluent we have identi ed neutrophil-derived miR-223 as a local biomarker of bacterial peritonitis,[8] and shown increased miR-21 in peritoneal brosis.[9]. We have identi ed panels of urinary miRNAs that predict delayed gra function following transplantation,[10] and detect diabetic kidney disease (DKD).[11] Our DKD patient data showed signi cantly decreased urinary miR-192 in those suffering from DKD,[11] supporting our previous ndings from renal biopsy analyses.[12]
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