Figure 3

Detection of differential miRNA processing and expression. Two sets of membranes were prepared as described in Figure 2. Each membrane was sequentially probed with labeled RNA marker (RNA M) for size standards, probe complementary to a specific miRNA that shows incomplete processing, a 21-nt region immediately next to the specific miRNA sequence, a second miRNA that shows complete processing and then U6 as a standard. MiR172 was used as a loading control. (a-b) The expression of miRC1 and miRC26, respectively, showing incomplete processing. Based on the expression of the control RNA miR172, the processing efficiency is calculated and presented below the blots. APE, Arbitrary Processing Efficiency (%) = Small Fragment Intensity (SFI)*100/[Sum of Large Fragment Intensity (LFI) + SFI]. RTA, Relative Transcription Activity = (LFI + SFI)/Control RNA Fragment Intensity (CFI). (c-d) The diagram of the specifically probed sequence regions is shown. (e-f) The expression of precursors only is shown of miRC1 and miRC26, respectively. (g-h) The expression of miRC2 and miRC27 respectively is shown to demonstrate complete processing detected on the same blot. For all the blots shown, L, leaf; F, flower; Fr-I, fruit at 19 Day After Bloom (DAB); Fr-II, fruit at 40 DAB; Fr-III, fruit at 82 DAB; R, root; B, bark.