Fig. 3: Quantification of endogenous miRNAs.
From: Quantification of purified endogenous miRNAs with high sensitivity and specificity
a Scheme to immobilize endogenous miRNAs. Total RNA is purified from cell lysate, tailed with poly(A) at the 3′-end, hybridized with complementary DNA strands, and immobilized on a surface using streptavidin–biotin interaction. b Single-molecule images of let-7a and let-7c miRNA from HeLa cell. c Sequential detection of endogenous let-7a and let-7c. Representative fluorescence intensity time traces identified as let-7a (top) and let-7c (bottom) are shown. Probes for let-7a detection were exchanged with probes for let-7c detection at 1200 s (dashed line). d Calibration for miRNA quantification. The number of spots identified as let-7a miRNA (red) and let-7c (gray) are plotted at varying combination of concentrations of target RNAs. The data are nicely fitted to a linear function (solid lines) with zero y-intercept and slopes of 2.8 spot/pM for let-7a and 2.7 spot/pM for let-7c (R2 = 0.998 for let-7a, and 0.996 for let-7c). e miRNA spike-in experiments. Varying amount of synthetic let-7a and let-7c miRNAs were spiked in total RNA of HeLa Cell, and their spot numbers were plotted as a function of their concentration. The data are nicely fitted to a linear function (solid lines) with the same slope (2.8 spot/pM), but different y-intercepts (R2 = 0.996 for let-7a, and 0.999 for let-7c). f let-7a detection in HeLa cell, let-7a overexpression HeLa cell, and mock HeLa cell. g Comparison of average copy numbers of let-7a and let-7c per 20 pg total RNA from HeLa cell, and human liver tissue. h Comparison of average copy numbers of let-7a and let-7c per 20 pg total RNA from MCF-7 exosome, and MCF-7 cell. To obtain each data point of d–h, three or more independent experiments were conducted. Data are mean ± SD. P values were determined by two-sided t test (****P < 0.0001). Source data are available in the Source data file.
