Effective knockdown with an artificial mirtron targeted against DMPK. HEK cells were transfected with 250 ng of psiCheck2.2 targets and 500 ng of mirtron and assayed 2 d later. (A,B) Fluorescence images and quantification of different DMPK mirtrons in a pEGFP-Mirt vector. (C) Knockdown of the luciferase target by DMPK mirtrons. (D) Sequence of DMPK-Mirt 5 and 13 and their corresponding shRNAs. (E,F) Luciferase target knockdown by DMPK-Mirt 5, DMPK5 shRNA after transfection with 250 ng of psiCheck2.2 and increasing amounts of mirtron/shRNA plasmid using the NAD/nonspecific shRNA plasmid to maintain total amount of plasmid transfected (500 ng in total). (G) Deep sequencing was performed with 18- to 25-nt RNA from HEK cells transfected with DMPK-Mirt 5 and 13. Sequences that were aligned to mirtron sequences were collected, 3′ tails were removed, and the sequences were aligned to chart the frequency of individual nucleotides appearing in the small RNA species harvested from the cells. (Red nucleotides) Active guide strand; (green nucleotides) flanking exonic sequences. (H) HEK cells were transfected with 500 ng of phDMPK and 500 ng of mirtron or NAD, and qPCR against hDMPK normalized to 18S RNA was performed on RNA harvested 2 d after. (*) p < 0.05; (**) p < 0.01; (***) p < 0.001 versus NAD/NS shRNA control.
