Centre for Molecular Medicine and Therapeutics, BC Children’s Hospital Research Institute; Department of Medical Genetics, University of British Columbia, Vancouver, BC, Canada
Centre for Molecular Medicine and Therapeutics, Vancouver, BC, Canada
Correspondence to: Michael R. Hayden, Centre for Molecular Medicine and Therapeutics, BC Children’s Hospital Research Institute, University of British Columbia, 3025-950 West 28th Ave, Vancouver, BC V5Z 4H4, Canada. Tel.:+1 604 875 3535; E-mail: [email protected].
Note:  These authors contributed equally to this work.
Note:  Current address: Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, Canada.
Abstract: Background:Therapeutics that lower mutant huntingtin (mHTT) have shown promise in preclinical studies and are in clinical development for the treatment of Huntington disease (HD). Multiple assays have been developed that either quantify mHTT or total HTT but may not accurately measure levels of wild type HTT (wtHTT) in biological samples. Objective:To optimize a method that can be used to resolve, quantify and directly compare levels of full length wtHTT and mHTT in HD samples. Methods:We provide a detailed quantitative immunoblotting protocol to reproducibly resolve full length wtHTT and mHTT in multiple HD mouse and patient samples. Results:We show that this assay can be modified, depending on the sample, to resolve wtHTT and mHTT with a wide range of polyglutamine length differences (ΔQs 22–179). We also demonstrate that this method can be used to quantify allele-selective lowering of mHTT using an antisense oligonucleotide in HD patient-derived cells. Conclusion:This quantitative immunoblotting method can be used to reliably resolve full-length HTT alleles with ΔQs≥22 and allows for direct comparison of wtHTT and mHTT levels in HD samples.