Identifying Circulating MiRNAs as Potential Biomarkers for Kidney Disease by qRT-PCR

Abstract

MicroRNAs (miRNAs) are short endogenous non-coding RNA molecules that play critical roles in gene regulation, cell differentiation, proliferation, apoptosis, and metabolism. MiRNA levels in blood and other body fluids are altered in many disease stages and may be potential biomarkers for diagnosis and prognosis. Evaluation of miRNA from these matrices is challenging due to the low abundance of nucleic acids which can impact extraction efficiency and reproducibility. To explore the utility of miRNAs as clinical biomarkers in human serum and urine, I developed an assay procedure using Trizol-LS for extractions of serum or urine in the presence of glycogen carrier. This extraction method resulted in the efficient and reproducible recovery of a spiked C. elegans miRNA control, cel-miR-39, as well as two endogenous miRNAs, hsa-miR-16 and hsa-miR-223, using specific Taqman miRNA Assays. With this optimized technique, a list of miRNA targets were obtained from a screening assay that compared serum and urine collected from healthy normal donors and patients with proteinuria or nephrotic syndromes. A few miRNA targets with the greatest alteration in expression level between normal and disease groups were further evaluated using individual Taqman Assays. MiR-146b appeared to be significantly down-regulated in serum from the disease group, while miR-21 seemed slightly up-regulated in nephrotic urine. Overall, the method developed in this thesis provides a robust approach to isolate and detect circulating miRNA in human serum and urine with high reproducibility. This study also suggests a list of miRNA candidates that are worth further investigation as potential non-invasive biomarkers for kidney related diseases.