(A) Sensitivity comparison between DIG-labeled splinted ligation and traditional DIG-labeled Northern blots with both assays using UV cross-linking: (lane 1) 60 ng of synthetic hsa-miR-21 was assayed by traditional DIG-labeled Northern blots, (lane 2) 30 ng of synthetic hsa-miR-21 was assayed by traditional DIG-labeled Northern blots, and (lane 3) 30 ng synthetic hsa-miR-21 was assayed using DIG-labeled splinted ligation. (B) Sensitivity comparison between EDC cross-linking (lane 1) and UV cross-linking (lane 2) based on splinted ligation. We used 20 fmol synthetic hsa-miR-21 for splinted ligation with an equal amount of sample (15 μL) loaded on each lane. All steps were the same except for the cross-linking step. (C–E) Assessment of the effect of splinted ligation and EDC cross-linking on DSLE and the comparison between DSLE and other methods using hsa-miR-21. (C) Quantification of the absolute detective sensitivity of DSLE using synthetic hsa-miR-21: (lane 1) 200 fmol, (lane 2) 40 fmol, (lane 3) 20 fmol, and (lane 4) 2 fmol. (D) Comparison of the sensitivity of DSLE and traditional DIG-labeled Northern blots on miR21 detection: 7 μg (lanes 1,4), 4 μg (lanes 2,5), and 2 μg (lanes 3,6) HeLa RNA were used for each assay. Photo-exposure time was 10 min. (E) The detection limit assay of DSLE using total RNA. The concentration of RNA was as follows: (lane 1) 0.78 μg, (lane 2) 0.39 μg, and (lane 3) 0.13 μg. (Lane M) 30 ng of synthetic hsa-miR-21 was detected by DSLE.
