MicroRNA isolation from urine: comparison of total RNA isolation (TRI) reagent and silica membrane-based extraction methods

Abstract

Background: microRNA (miRNA) extraction from urine samples is challenging, especially for sensitive downstream analyses such as quantitative reverse transcription polymerase chain reaction (RT-qPCR). While commercial kits are available, their high cost can be prohibitive for repetitive experimental studies. In this context, this study aimed to compare the RNA yield and purity scores of conventional Total RNA Isolation (TRI) reagent-based to silica membrane-based extraction method.

Methods: miRNA was extracted from urine samples using three methods: 1) Silica membrane-based (Method 1); 2) TRI reagent-based (Method 2); and 3) Improvised TRI reagent-based (Method 3). RNA yield and purity score (A260/280 and A260/230) were analysed using one-way repeated measures ANOVA. The relative expression of hsa-miR-21-5p normalized to RNU6B was assessed by RT-qPCR for extracts from Method 1 and 2 to compare the effects of silica membrane-based and TRI reagent-based.

Results: Total RNA yield (p-value = 0.005) and A260/230 (p-value < 0.001) differed significantly between all methods except for the A260/280 (p-value = 0.177). Specifically, higher RNA yield and A_260/230 from Method 3 were noted compared to Method 1 (p-value = 0.007 and 0.010) and Method 2 (p-value = 0.046 and 0.009), with A260/230 remaining outside the acceptable range. The total RNA yield, A260/280 and A260/230, is not significantly different between Methods 1 and 2 (p-value = 0.291, 0.566, and 1.000).

Conclusion: While the purity could be improved, TRI reagent method shows promise as an alternative for high-yield RNA extraction. It is particularly useful for repetitive downstream experiments in research and clinical applications.

Keywords: RNA extraction; RT-qPCR; Silica membrane; TRI reagent; Urine; miRNA; microRNA.