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. 2017 Sep 6;45(15):e144.
doi: 10.1093/nar/gkx588.

Two-tailed RT-qPCR: a novel method for highly accurate miRNA quantification

Peter Androvic  1   2 , Lukas Valihrach  1 , Julie Elling  3 , Robert Sjoback  3 , Mikael Kubista  1   3
Affiliations

Two-tailed RT-qPCR: a novel method for highly accurate miRNA quantification

Peter Androvic et al. Nucleic Acids Res. .

Abstract

MicroRNAs are a class of small non-coding RNAs that serve as important regulators of gene expression at the posttranscriptional level. They are stable in body fluids and pose great potential to serve as biomarkers. Here, we present a highly specific, sensitive and cost-effective system to quantify miRNA expression based on two-step RT-qPCR with SYBR-green detection chemistry called Two-tailed RT-qPCR. It takes advantage of novel, target-specific primers for reverse transcription composed of two hemiprobes complementary to two different parts of the targeted miRNA, connected by a hairpin structure. The introduction of a second probe ensures high sensitivity and enables discrimination of highly homologous miRNAs irrespectively of the position of the mismatched nucleotide. Two-tailed RT-qPCR has a dynamic range of seven logs and a sensitivity sufficient to detect down to ten target miRNA molecules. It is capable to capture the full isomiR repertoire, leading to accurate representation of the complete miRNA content in a sample. The reverse transcription step can be multiplexed and the miRNA profiles measured with Two-tailed RT-qPCR show excellent correlation with the industry standard TaqMan miRNA assays (r2 = 0.985). Moreover, Two-tailed RT-qPCR allows for rapid testing with a total analysis time of less than 2.5 hours.

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Figures

Figure 1.
Figure 1.
Schematic of Two-tailed RT-qPCR. (A) Two-tailed RT primer having two hemiprobes connected by a hairpin folding sequence. (B) The hemiprobes bind cooperatively, one at each end of the target miRNA, forming a stable complex. (C) Reverse transcriptase binds the 3′-end of the hybridized Two-tailed RT primer and elongates it to form tailed cDNA. (D) The cDNA is amplified by qPCR using two target-specific primers.
Figure 2.
Figure 2.
Importance of the 5′-hemiprobe for sensitivity and specificity. (A) Two-tailed RT primers with 5 nt complementary 3′-hemiprobe and either 10 nt complementary 5′-hemiprobe (top), 10 nt non-complementary 5′-hemiprobe (middle), or no 5′-hemiprobe at all (bottom) targeting let-7a. Cq values obtained with the Two-tailed RT primer having a complementary 5′-hemiprobe are about nine cycles lower than those obtained with the Two-tailed RT primers lacking 5′-complementarity. (B) Two-tailed RT primers used to assay targets that differ in one nucleotide. The variable nucleotide is in the non-interrogated region between the hybridization sites of the 3′- and 5′-hemiprobes (left) and the variable nucleotide is within the 5′-hemiprobe binding site (right).
Figure 3.
Figure 3.
Dynamic range and sensitivity of Two-tailed RT-qPCR, TaqMan, Quanta and miQPCR. (A) Amplification plots and standard curves of let-7d assayed in water and against a background of 100 ng yeast RNA. The dynamic range is seven logs. (B) Standard curves of let-7a, let-7d, and miR-21 assayed with Two-tailed RT-qPCR, TaqMan, Quanta, and miQPCR. Cq values outside the linear range are indicated with red border.
Figure 4.
Figure 4.
Specificity of Two-tailed RT-qPCR, TaqMan, Quanta, and miQPCR. (A) Measured false-positive levels of let-7 miRNA family members expressed relative to the level of the targeted member. (B) Sequences of eight members of the let-7 family. Nucleotide variations relative to let-7a are indicated. (C) Cq values and relative detection levels of pre-miRNAs relative to the targeted mature microRNA measured with Two-tailed RT-qPCR.
Figure 5.
Figure 5.
Comparison of expression profiles measured with Two-tailed RT-qPCR and TaqMan miRNA assays. (A) Relative fold changes of the expression of each target in seven tissues measured with Two-tailed RT-qPCR and TaqMan miRNA assays, respectively. (B) Overall correlation of the relative expression changes measured with the two methods.
Figure 6.
Figure 6.
Comparison of singleplex and multiplex Two-tailed RT-qPCR. (A) ΔCq = Cqmultiplex - Cqsingleplex. (B) Overall correlation of the relative expression changes between tissues measured with the singleplex and multiplex protocol.
Figure 7.
Figure 7.
Relative sensitivities of Two-tailed RT-qPCR, TaqMan, Quanta, and miQPCR to miR-21 isomiRs. Cq values are normalized such that the Cq of the canonical form is set to 20. Error bars indicate SD of two independent cDNA syntheses.
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