Fig. 6
Differentiation of isomiRs from their canonical forms by qRT-PCR. a selection considerations. b Schematic diagram of assay design. Exiqon’s miRNA qRT-PCR assay involves polyadenylation at the 3′ end, rendering the assay high 3′ end specificity. Through optimizing qPCR conditions, they can differentiate 3′ isomiR from canonical forms. c Example on qPCR condition optimization: PCR annealing temperature (Ta) testing for maximal differentiation between canonical form (hsa-miR-200b-3p) and its isomiR (3′ addition C form). c i Amplification of canonical form oligos (104, 103, 102 copies; blue curves) and 3′ addition C form oligos (104, 103, 102 copies; red curves) by assay targeting canonical form (left panel), and assay targeting 3′ addition C form (right panel), respectively, under various Ta (PCR profile refers to Additional file 2: Figure S1); c ii Summary of Ta testing, bar charts represent relative detection of canonical form oligos (red) and 3′ addition C form oligos (blue). Data represent mean ± s.d. of three data points based on the amplification of 104, 103, and 102 copies of oligos