Fig 1.
Schematic of the pincer probe-based miRNA quantification system.
The pincer probe-based miRNA quantification assay included two steps: A) pincer probe reverse transcription and B) TaqMan real-time PCR. First, the pincer probe captured the target miRNA by undergoing a “pincer-like movement” and initiated transcription using the miRNA as a primer and the pincer probe as a template (A1). Although they can bind to the pincer probe, the nonmature miRNAs cannot initiate reverse transcription because of the 3′ end overhang (A1*). Then, the reverse transcriptase displaced the miRNA sequence complementary to the 5′ arm of the pincer probe that was encountered during synthesis and added a ciRNA tag to the 3′ end of the pincer probe (A2). Finally, the ciRNA-tagged pincer probes were quantified using conventional TaqMan qPCR assays that include the ciRNA-specific forward primer, the reverse primer, and dye-labeled TaqMan probes (B).
Fig 2.
Testing the miRNA quantification system using pincer probes and controls.
The lane designated “M” represents the size maker with an upper band of 200 bp and a lower band of 100 bp. The perfectly matched pincer probe and its target miRNA were actively reverse transcribed and were ciRNA tagged (lanes A1 to A5). The total amounts of miRNA in lanes A1 to A5 were 0.5, 1, 2, 4, and 8 pmol, respectively. The quantified amounts for bands A1 to A5 were 335.06, 662.25, 676.20, 1,345.30, and 2,547.50, respectively (Gel-Pro Analyzer). The mutations in the control probes were located in the middle of the arms (Ctr-A in the left arm and Ctr-B in the right arm) or in the 3′ cohesive end of the miRNA/pincer-probe duplex (Ctr-C in the pincer probe and Ctr-D in miRNA). Reverse transcription of miRNA with pincer-probe controls A, C and D (lanes B, D and E) did not produce correct products required for miRNA quantification. The pincer-probe control B (lane C) produced observable signal indicated that the assay may less to be precise if the mutation is close to the 5′ end. The reverse transcription product was detected using chemiluminescent photography.
Fig 3.
Dynamic range, specificity, and sensitivity of the pincer probe-based miRNA quantification assay.
Plot A shows the correlation between the miRNA input and the Cq (threshold of cycle) values in the test assays which in the absence or presence of total RNA (Spike-In test). Plots B and C show the Cq values from the test assays using perfectly matched pincer probes or control pincer probes. The synthetic miRNA input ranged from 1.0 X 10-3 fM (about 60 copies per reaction) to 1.0 X 103 fM in the PCR (lane A to G, perfectly matched pincer probes). The amount of synthetic miRNA for the four pincer-probe control test assays (Ctr-A to Ctr-D) was 10 pmol. The total RNA input for the Spike-In test assay was 1 μg. The amplification chart and raw data are available in S1 File.
Fig 4.
Quantification of miR-133a, miR-122a, and miR-155 expression in different tissues.
A bar graph shows the normalized expression value detected by qPCR for each miRNA, and an autoradiogram shows the average signal of Northern blot for three repeated experiments. In the bar graph, the signal from nine tissues was normalized to the internal control of Met-tRNA. The background signal obtained from the sample containing H2O instead of RNA was set to zero.
Table 1.
Multiplex detection of miRNAs using pincer probe or stem-loop real-time RT-PCR.
Table 2.
Discrimination between the mature and the precursor miRNAs using pincer probe or linear real-time RT-PCR.
Table 3.
Cross reaction (%) of each let-7 miRNAs by specific pincer probe real-time PCR assays.