Figure 1.
Diagram of USLP method on screening miRs.
Light blue lines indicate total RNA. Arrowheads indicate directions of polymerization. 8 random sequences at the 3′ end of stem-loop primer (red) can bind to all mature miRs. In the reaction, mature miR was reversely transcribed into cDNA under the reaction conditions of RT; then, miR was screened and quantified by fluorescence quantitative PCR using miR specific forward primer and universal reverse primer. The forward PCR primer adds additional length with nucleotides that optimize its melting temperature (Tm) and enhance assay specificity. A universal reverse primer that is complementary to a sequence within the RT stem-loop primer.
Figure 2.
Optimization of qPCR for USLP concentration.
USLP at the concentration of 1 µmol/L had higher detection efficiency than others.
Figure 3.
Dynamic range and sensitivity analysis of USLP for measurement of synthetic miR-155.
Synthetic miR-155 was diluted by more than seven orders of magnitude from 103 copies/µl to 109 copies/µl.
Figure 4.
Specificity evaluation of the TSLP and USLP.
(A) PCR bands of miR-150 and miR-155 were detected by both TSLP and USLP methods. The PCR products were run on 3% agarose gel in 1X TBE stained with ethidium bromide. (B) Melting curve of the TSLP and USLP methods for detecting synthetic miR-155.
Table 1.
The detection of different concentrations of miR-155 in USLP method.
Figure 5.
Standard curve of the TSLP and USLP methods for measurement of miR-155.
Curve of the new method assay was a straight line (R2 = 0.993) with a slope of 3.63 (PCR efficiency = 88.6%) over seven orders of magnitude of Synthetic miR-155. Curve of TSLP method was also a straight line (R2 = 0.988) with slope of 3.16 (PCR efficiency = 107.2%) over seven orders of magnitude of Synthetic miR-155.
Figure 6.
Synthetic miR-155 was diluted by over four orders of magnitude and qRT-PCR using the TSLP and USLP methods.
Figure 7.
Expression levels of miR-150 and miR-155 in T lymphocytes by using the TSLP and USLP.
Expression of miR-150 was decreased nearly 10-fold with prolonged incubation time (incubated at 0, 24, 48, and 72 h). Meanwhile, the expression of miR-155 was increased more than 7-fold with prolonged incubation time. The results of the TSLP and USLP methods were basically consistent in the expression of miR-150 and miR-155. *P<0.05, **P<0.01 and ***P<0.001 vs 0 h; #P<0.05, ##P<0.01 and ###P<0.001 vs control group.