MicroRNA Detection Using a Double Molecular Beacon Approach: Distinguishing Between miRNA and Pre-miRNA

Abstract

MicroRNAs (miRNAs) are small, noncoding RNAs that post-transcriptionally regulate gene expression and are recognized for their roles both as modulators of disease progression and as biomarkers of disease activity, including neurological diseases, cancer, and cardiovascular disease (CVD). Commonly, miRNA abundance is assessed using quantitative real-time PCR (qRT-PCR), however, qRT-PCR for miRNA can be labor intensive, time consuming, and may lack specificity for detection of mature versus precursor forms of miRNA. Here, we describe a novel double molecular beacon approach to miRNA assessment that can distinguish and quantify mature versus precursor forms of miRNA in a single assay, an essential feature for use of miRNAs as biomarkers for disease. Using this approach, we found that molecular beacons with DNA or combined locked nucleic acid (LNA)-DNA backbones can detect mature and precursor miRNAs (pre-miRNAs) of low (< 1 nM) abundance in vitro. The double molecular beacon assay was accurate in assessing miRNA abundance in a sample containing a mixed population of mature and precursor miRNAs. In contrast, qRT-PCR and the single molecular beacon assay overestimated miRNA abundance. Additionally, the double molecular beacon assay was less labor intensive than traditional qRT-PCR and had 10-25% increased specificity. Our data suggest that the double molecular beacon-based approach is more precise and specific than previous methods, and has the promise of being the standard for assessing miRNA levels in biological samples.

Keywords: PCR.; cardiovascular disease; molecular beacon, microRNA, microRNA detection.

Figure 1

Schematics of Molecular Beacon Hybridization to target mature and precursor miRNA. In the absence of complementary target, molecular beacons form a stem-loop structure that brings the quencher in close proximity to the fluorophore, thereby quenching the fluorescence emission. Hybridization of the molecular beacons to its target mature or precursor miRNA opens the molecular beacon hairpin, thereby leading to physical separation of the fluorophore from the quencher and allowing fluorescence to be measured upon excitation. (A) Hybridization of molecular beacons to the mature miRNA target sequence and unintended hybridization of the mature molecular beacon to pre-miRNA. (B) Hybridization of pre-miRNA molecular beacons to the pre-miRNA loop sequence. Also depicted is the inability for mature miRNA to hybridize to the pre-miRNA molecular beacon, though 2-3 nucleotide bases from the pre-miRNA molecular beacon may hybridize to the mature miRNA. However, in this latter example there is not enough hybridization to separate the quencher from the fluorophore. The use of LNA bases allows the pre-miRNA molecular beacon to have a relatively higher affinity for its intended pre-miRNA target then to complementary mature miRNA sequences.

Figure 2

Determining Molecular Beacon Sensitivity for each Target. Differences in hybridization of molecular beacons to target miRNA with varying backbones consisting of DNA and LNA/DNA bases. Molecular beacons (200 nM) were incubated with each synthetic target separately (pre-miR-21 or miR-21 [A] and pre-miR-27b or miR-27b [B] [0-5 nM]) in PBS at 37°C for 60 min prior to recording the fluorescence intensity; three pre-miRNA LNA/DNA molecular beacons were designed and tested while previously verified mature miRNA DNA molecular beacons were tested for sensitivity only. For each experiment, background fluorescence was subtracted from signal produced by hybridization of the molecular beacon to its target miRNA.

Figure 3

Molecular Beacon Hybridization to Targets in Presence of Oligo Blockers. The molecular beacons were designed to hybridize mature miRNA targets. The presence of oligo blockers (complementary or competitive) demonstrated the specificity of each molecular beacon. Mature miRNA molecular beacons targeting miR-21(A) and -27b (B) were incubated with mixtures of synthetic mature and pre-miRNA at varying concentrations (0-200nM). The design of each mature miRNA molecular beacon allowed for the unintended hybridization to precursor miRNA. Therefore, the presence of a mature miRNA blocker inhibited signal from the molecular beacon while the presence of pre-miRNA blocker showed a modest change in fluorescence intensity. Each bar represents mean ± SE of 3-6 separate experiments.

Figure 4

Double Molecular Beacon Assay. Hybridization of molecular beacons to mature or precursor miRNA. The mature miRNA molecular beacon (200 nM) and pre-miRNA molecular beacon (200 nM) (A: miR-21; B: miR-27b) were incubated with mixtures of mature and pre-miRNA targets at the indicated concentrations (60 min at 37°C). The relative fluorescence signal from each molecular beacon alone in PBS was used as a background measurement and subtracted from the sample signal. Each data point represents mean ± SEM of three separate experiments.

Figure 5

Comparison of Double Molecular Beacon Assay to qRT PCR and Single Molecular Beacon Assay. Comparison of double molecular beacon assay with qRT-PCR and single molecular beacon assay in simultaneously measurement of mature and precursor miRNAs. Assays were performed on solutions containing mixed, equimolar concentrations (A. 10 nM and B. 50 nM) of mature and precursor forms of miR-21 and miR-27b. For single and double MB assays, background fluorescence was subtracted from hybridized beacon signal. For qRT-PCR standard TaqMan protocol was followed. Standard concentration curves were used to determine concentration in all methods. Each data point represents mean ± SD of six separate experiments. Tables display the numerical concentrations and the P-values (paired t-tests P > 0.05) (significance indicated in red).

Figure 5

Comparison of Double Molecular Beacon Assay to qRT PCR and Single Molecular Beacon Assay. Comparison of double molecular beacon assay with qRT-PCR and single molecular beacon assay in simultaneously measurement of mature and precursor miRNAs. Assays were performed on solutions containing mixed, equimolar concentrations (A. 10 nM and B. 50 nM) of mature and precursor forms of miR-21 and miR-27b. For single and double MB assays, background fluorescence was subtracted from hybridized beacon signal. For qRT-PCR standard TaqMan protocol was followed. Standard concentration curves were used to determine concentration in all methods. Each data point represents mean ± SD of six separate experiments. Tables display the numerical concentrations and the P-values (paired t-tests P > 0.05) (significance indicated in red).

Figure 6

Comparison of qRT-PCR and Double Molecular Beacon Assay in assessing mature and precursor miRNA levels in total cellular RNA. Comparison of qRT-PCR and double molecular beacon assay in assessing miRNA concentrations in total RNA isolated from HAECs. MiRNA levels (A. miR-21 and B. miR-27b) in total RNA extracted from HAECs was determined. For each qRT-PCR reaction, the standard TaqMan protocol was used using 1 µg of total RNA. For the double molecular beacon hybridization assays, 200 nM of each molecular beacon and 1 µg of total RNA were used. Standard concentration curves were used to determine concentration in both methods. Additionally, for the double molecular beacon assay background fluorescence was subtracted from beacon signal. Each data point represents Mean ± SD of six separate experiments. C. Table displaying the numerical concentrations and the p-values (paired t-tests). (P > 0.05).

Figure 7

Comparison of qRT-PCR and Double Molecular Beacon Assay in assessing mature and precursor miRNA levels in whole blood RNA. Comparison of qRT-PCR and double molecular beacon assay in assessing miR-21 concentrations in total RNA isolated from whole blood (obtained from healthy volunteer). MiRNA levels (A. miR-21) in total RNA extracted from whole blood was determined. For each qRT-PCR reaction, the standard TaqMan protocol was used with 3 µg of total RNA. For the double molecular beacon hybridization assays, 500 nM of each molecular beacon and 3 µg of total RNA were used. Standard concentration curves were used to determine concentration in both methods. Additionally, for the double molecular beacon assay background fluorescence was subtracted from beacon signal. Each data point represents Mean ± SD of six separate experiments. B. Table displaying the numerical concentrations and the p-values (paired t-tests) (P > 0.05).