Although a variety of qPCR-based analysis solutions are currently available to quantify miRNAs, they may lack either sensitivity or specificity, have cumbersome workflows, or have limited instrument and software compatibility. To address these challenges, we have developed an miRNA analysis workflow that includes both a universal RT step and the superior sensitivity and specificity of TaqMan probe-based chemistry.
miRNAs can have extensive sequence homology and are grouped in highly conserved families of nearly identical sequence. Cross-reactivity of TaqMan Advanced miRNA Assays was tested between four hsa-let-7 miRNAs (Table 1).
TaqMan Advanced miRNA Assays exhibited zero cross-reactivity for the majority of target combinations. For hsa-let-7c that differs from hsalet-7a by a single base (G/A), results show less than 2% reactivity. Thus, TaqMan Advanced miRNA Assays are capable of discriminating closely related miRNA sequences, differing by as little as a single nucleotide.
Table 1. Percent reactivity of TaqMan Advanced miRNA Assays tested against each hsa-let-7 family member and calculated based on exact-match results.
The TaqMan Advanced miRNA Assay workflow incorporates a unique miR-Amp universal amplification step, combined with proven TaqMan Assay design and chemistry, to enable consistent and reliable results down to as little as 1 pg of RNA. This level of sensitivity is required for challenging investigations such as the study of noninvasive biomarkers where circulating miRNA levels are limited.
To assess limits of detection, Invitrogen Human Liver Total RNA was diluted and 10 ng, 1 ng, 100 pg, 10 pg, and 1 pg were tested with four assays chosen to represent a range of expression levels in liver (Figure 1). TaqMan Advanced miRNA Assays and Exiqon miRCURY™ LNA™ microRNA qPCR assays were compared in parallel. The experiments were carried out according to manufacturer’s recommended protocol including amount of total RNA input.
Across each assay and every dilution tested in this study, TaqMan Advanced miRNA Assays exhibited lower Ct values, allowing for overall greater sensitivity compared with the equivalent miRCURY LNA microRNA assay. Furthermore, for several dilutions (100 pg, 10 pg, and 1 pg) the miRCURY LNA hsa-miR-141-3p assay failed to amplify, and the 1 pg sample failed for the miRCURY LNA hsa-miR-16 assay.
Figure 1. Amplification of serially diluted total RNA from human liver. Across all four assays chosen to represent high, medium, and low relative target abundance, higher sensitivity was obtained with TaqMan Advanced miRNA Assays compared with miRCURY LNA microRNA assays. Variability is also limited across all TaqMan Advanced miRNA Assays, with error bars representing standard deviations calculated from triplicate reactions.
In addition, several serum plasma panels were tested to compare performance by number of miRNAs detected, including TaqMan Advanced miRNA Array – Serum/Plasma, Exiqon Serum/Plasma Focus miRNA PCR Panel V4, and Qiagen miScript miRNA PCR Array – Human Serum and Plasma. The TaqMan Advanced miRNA Array detected more targets in serum/plasma samples (Figure 2).
To determine accuracy of expression, serum samples 1 and 2 were generated by spiking three different synthetic miRNA targets (hsa-miR-10a, hsa-miR-7a, and hsa-miR-302a) into the samples (Table 2). Accuracy was determined by comparing expected and observed concentration differences between serum sample 1 and 2 for all three miRNAs studied. TaqMan Advanced miRNA Assays more accurately quantified small differences in miRNA concentrations when compared to miRCURY LNA microRNA assays.
|TaqMan Advanced miRNA Assays||2.4||0.8||-1.2|
|miRCURY LNA micoRNA assays||2.9||0||-0.5|
Table 2. Relative mean ΔCt values for synthetic miRNAs spiked into serum. Serum sample 2 was spiked with varying concentrations of three synthetic miRNAs, while serum sample 1 was spiked with fixed concentrations of the same miRNAs. The miRNAs were quantified by RT-qPCR and concentration differences expressed as relative expression (log2) values.
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For Research Use Only. Not for use in diagnostic procedures.