TY - JOUR
T1 - A multicenter comparison of quantification methods for antisense oligonucleotide-induced DMD exon 51 skipping in Duchenne muscular dystrophy cell cultures
AU - Hiller, Monika
AU - Falzarano, Maria Sofia
AU - Garcia-Jimenez, Iker
AU - Sardone, Valentina
AU - Verheul, Ruurd C
AU - Popplewell, Linda
AU - Anthony, Karen
AU - Ruiz-Del-Yerro, Estibaliz
AU - Osman, Hana
AU - Goeman, Jelle J
AU - Mamchaoui, Kamel
AU - Dickson, George
AU - Ferlini, Alessandra
AU - Muntoni, Francesco
AU - Aartsma-Rus, Annemieke
AU - Arechavala-Gomeza, Virginia
AU - Datson, Nicole A
AU - Spitali, Pietro
PY - 2018/10/2
Y1 - 2018/10/2
N2 - Background: Duchenne muscular dystrophy is a lethal disease caused by lack of dystrophin. Skipping of exons adjacent to out-of-frame deletions has proven to restore dystrophin expression in Duchenne patients. Exon 51 has been the most studied target in both preclinical and clinical settings and the availability of standardized procedures to quantify exon skipping would be advantageous for the evaluation of preclinical and clinical data. Objective: To compare methods currently used to quantify antisense oligonucleotide–induced exon 51 skipping in the DMD transcript and to provide guidance about the method to use. Methods: Six laboratories shared blinded RNA samples from Duchenne patient-derived muscle cells treated with different amounts of exon 51 targeting antisense oligonucleotide. Exon 51 skipping levels were quantified using five different techniques: digital droplet PCR, single PCR assessed with Agilent bioanalyzer, nested PCR with agarose gel image analysis by either ImageJ or GeneTools software and quantitative real-time PCR. Results: Differences in mean exon skipping levels and dispersion around the mean were observed across the different techniques. Results obtained by digital droplet PCR were reproducible and showed the smallest dispersion. Exon skipping quantification with the other methods showed overestimation of exon skipping or high data variation. Conclusions: Our results suggest that digital droplet PCR was the most precise and quantitative method. The quantification of exon 51 skipping by Agilent bioanalyzer after a single round of PCR was the second-best choice with a 2.3-fold overestimation of exon 51 skipping levels compared to digital droplet PCR.
AB - Background: Duchenne muscular dystrophy is a lethal disease caused by lack of dystrophin. Skipping of exons adjacent to out-of-frame deletions has proven to restore dystrophin expression in Duchenne patients. Exon 51 has been the most studied target in both preclinical and clinical settings and the availability of standardized procedures to quantify exon skipping would be advantageous for the evaluation of preclinical and clinical data. Objective: To compare methods currently used to quantify antisense oligonucleotide–induced exon 51 skipping in the DMD transcript and to provide guidance about the method to use. Methods: Six laboratories shared blinded RNA samples from Duchenne patient-derived muscle cells treated with different amounts of exon 51 targeting antisense oligonucleotide. Exon 51 skipping levels were quantified using five different techniques: digital droplet PCR, single PCR assessed with Agilent bioanalyzer, nested PCR with agarose gel image analysis by either ImageJ or GeneTools software and quantitative real-time PCR. Results: Differences in mean exon skipping levels and dispersion around the mean were observed across the different techniques. Results obtained by digital droplet PCR were reproducible and showed the smallest dispersion. Exon skipping quantification with the other methods showed overestimation of exon skipping or high data variation. Conclusions: Our results suggest that digital droplet PCR was the most precise and quantitative method. The quantification of exon 51 skipping by Agilent bioanalyzer after a single round of PCR was the second-best choice with a 2.3-fold overestimation of exon 51 skipping levels compared to digital droplet PCR.
UR - http://www.mendeley.com/research/multicenter-comparison-quantification-methods-antisense-oligonucleotideinduced-dmd-exon-51-skipping
U2 - 10.1371/journal.pone.0204485
DO - 10.1371/journal.pone.0204485
M3 - Article
SN - 1932-6203
VL - 13
SP - 1
EP - 15
JO - PLoS ONE
JF - PLoS ONE
IS - 10
ER -