Detection of miR-133a-5p Using a Molecular Beacon Probe for Investigating Postmortem Intervals

Abstract

Background: When a body is discovered at a crime or murder scene, it is crucial to examine the body and estimate its postmortem interval (PMI). Accurate estimation of PMI is vital for identifying suspects and providing clues to resolve the case. MicroRNAs (miRNAs or miRs) are small non-coding RNAs that remain relatively stable in the cell nucleus even after death-related changes occur. Objective: This study developed a molecular beacon probe for mmu-miR-133a-5p and assessed its use in mouse muscle tissue at temperatures of 4 °C and 21 °C to estimate the PMI. Methods: A total of 36 healthy adult male BALB/c mice were divided into 9 PMI time points (0, 2, 6, 8, and 10 days) with 3 mice per time point, and they were exposed to 4 °C and 21 °C. Next, the expression pattern of mmu-miR-133a in the skeletal muscle tissue over a 10-day PMI period was analyzed using the developed molecular beacon probe. Results: The molecular beacon (MB) probe was designed for optimal thermodynamic stability with a hairpin structure that opened in the presence of mmu-miR-133a-5p, thus separating the fluorophore from the quencher and resulting in a strong fluorescence signal at 495 nm. Fluorescence intensity increased with mmu-miR-133a-5p concentration from 1 ng/μL to 1000 ng/μL and exhibited a strong correlation (R² = 0.9966) and a detection limit of 1 ng/μL. Subsequently, the expression level of mmu-miR-133a-5p was observed to be stable in mouse skeletal muscle tissue at both 4 °C and 21 °C. Conclusions: This user-friendly assay can complete measurements in just 30 min after RNA extraction and is suitable for point-of-care testing, and it possesses the potential to improve existing complex and time-consuming methods for PMI estimation.

Keywords: biomarker; microRNA; molecular beacon probe; postmortem interval.

Figure 1

Schematic illustration for detection of mmu-miR-133a-5p using MB to assess PMI. (A) Thirty-six healthy adult male BALB/c mice were randomly divided into six groups based on different PMI intervals (0, 1, 2, 6, 8, and 10 days), with each group containing three mice. The mice were exposed to two temperature conditions (4 °C and 21 °C). Skeletal muscle tissues were collected from each mouse, homogenized with TRIzol reagent, and total RNA was extracted. (B) The MB probe was engineered for optimal thermodynamic stability in its hairpin structure, but it unfolds in the presence of mmu-miR-133a. (C) Steps of the MB probe assay for quantifying mmu-miR-133a. Created in Biorender.

Figure 2

Analytical performance of molecular beacon probe assay for mmu-miR-133a-5p. (A) Melt curve of the molecular beacon probe for mmu-miR-133a-5p. (B) The fluorescence spectra of the molecular beacon probe in response to varying concentrations of synthetic mmu-miR-133a. (C) The standard curve between fluorescence intensity versus the target miRNA concentration. (D) Detection limits of the molecular beacon probe assay for mmu-miR-133a-5p using 10-fold serial dilutions of synthetic miR-133a-5p (from 1 ng/μL to 1000 ng/μL). The dotted line represents the limit of detection.

Figure 3

Specificity of the molecular beacon probe assay for mmu-miR-133a-5p. (A) A graphical comparison of wild-type and mutant miR-133a-5p used to assess the specificity of the molecular beacon probe assay for mmu-miR-133a-5p. (B) The fluorescence spectra of the molecular beacon probe in reactions with wild-type and mutant miR-133a-5p.

Figure 4

Detection of mmu-miR-133a-5p using the MB probe at different time intervals at (A) 4 °C or (B) 21 °C in mouse skeletal muscle. Data are presented as mean ± standard error of the mean.