Comparison of Fecal MicroRNA Isolation Using Various Total RNA Isolation Kits

Abstract

Fecal specimens have long been regarded as promising sources for gastrointestinal cancer screening and have, thus, been extensively investigated in biomarker research. MicroRNAs (miRNAs) are small, non-coding RNA molecules involved in regulating various biological processes. They are commonly dysregulated during tumor development and exhibit differential expression in feces. To assess the preanalytical feasibility of fecal miRNA analysis, we systematically compared the performance of commonly used total RNA extraction methods. Fecal samples from healthy subjects were utilized for this evaluation. Various methods, including miRNeasy, Universal, Trizol, RNeasy, and mirVana kits, were employed to isolate total RNA. MiRNA expression analyses were conducted using TaqMan or SYBR Green qRT-PCR for a subset of miRNAs, with externally spiked-in cel-miR-39 used for normalization. Most methods demonstrated similar performance in terms of the total RNA concentration and purity. Externally spiked cel-miR-39 and endogenous miRNAs (RNU6b, miR-16, and miR-21) exhibited comparable concentrations across the different RNA isolation methods, whereas the RNeasy mini kit consistently yielded lower values. Our findings suggest that various isolation methods produce reproducible and comparable miRNA expression results, supporting the potential comparability and translational applicability of miRNA-based biomarker research in the future.

Keywords: fecal biomarker; microRNA; microRNA kits; total RNA.

Figure 1

Graphical study design. Workflow of the study.

Figure 2

Total RNA and purity. (A) General comparison on total RNA level. (B) Comparison of purity of miRNAs. ** p ≤ 0.01; *** p ≤ 0.001.

Figure 3

Correlation between the kits. Pearson correlation between the different kits based on Ct values and the amount of total RNA and RNA purity. ** p = 0.01; **** p = <0.0001.

Figure 4

Performance of miRNA analysis and reproducibility analyses. (A) Data are presented as raw Ct for RNU6b and cel-miR-39, miR-16, and miR-21. (B) Data forming direct comparisons between miRNeasy and mirVana kits are presented as raw Ct for cel-miR-39, miR-16, and miR-21. (C) Extraction analyses for the independent qPCR run to confirm the validity of the data. ** p ≤ 0.01; *** p ≤ 0.001.

Figure 5

Comparison of normalized miR-21 levels. (A) miR-21 expression normalized to cel-miR-39 ΔCt method. (B) miR-21 expression normalized to combined cel-miR-39 and miR-16.