title: The influence of storage parameters on measurement of survival motor neuron (SMN) protein levels: implications for pre-clinical studies and clinical trials for spinal muscular atrophy.
abstract: Spinal muscular atrophy (SMA) is caused by low levels of survival motor neuron (SMN) protein. A growing number of potential therapeutic strategies for SMA are entering pre-clinical and clinical testing, including gene therapy and antisense oligonucleotide-based approaches. For many such studies SMN protein levels are used as one major readout of treatment efficacy, often necessitating comparisons between samples obtained at different times and/or using different protocols. Whether differences in tissue sampling strategies or storage parameters have an influence on measurable SMN levels remains to be determined. We assessed murine SMN protein immunoreactivity over time and under differing tissue storage conditions. SMN protein levels, measured using sensitive quantitative fluorescent western blotting, declined rapidly over a period of several days following sample collection, especially when protein was extracted immediately and stored at -20°C. Storage of samples at lower temperatures (-80°C), and as intact tissue, led to significantly better preservation of SMN immunoreactivity. However, considerable deterioration in measurable SMN levels occurred, even under optimal storage conditions. These issues need to be taken into consideration when designing and interpreting pre-clinical and clinical SMA studies where SMN protein levels are being measured.
authors.affiliation: Euan MacDonald Centre for Motor Neurone Disease Research, University of Edinburgh, Edinburgh, EH16 4SB, UK; Centre for Integrative Physiology, University of Edinburgh, Edinburgh EH8 9XD, UK.
authors.forename: Gillian
authors.initials: G
authors.lastname: Hunter
authors.affiliation: Euan MacDonald Centre for Motor Neurone Disease Research, University of Edinburgh, Edinburgh, EH16 4SB, UK; Centre for Integrative Physiology, University of Edinburgh, Edinburgh EH8 9XD, UK.
authors.forename: Sarah L
authors.initials: SL
authors.lastname: Roche
authors.affiliation: Euan MacDonald Centre for Motor Neurone Disease Research, University of Edinburgh, Edinburgh, EH16 4SB, UK; Centre for Integrative Physiology, University of Edinburgh, Edinburgh EH8 9XD, UK.
authors.forename: Eilidh
authors.initials: E
authors.lastname: Somers
authors.affiliation: Wolfson Centre for Inherited Neuromuscular Disease, RJAH Orthopaedic Hospital, Oswestry, UK.
authors.forename: Heidi R
authors.initials: HR
authors.lastname: Fuller
authors.affiliation: Euan MacDonald Centre for Motor Neurone Disease Research, University of Edinburgh, Edinburgh, EH16 4SB, UK; Centre for Integrative Physiology, University of Edinburgh, Edinburgh EH8 9XD, UK; Institute for Science and Technology in Medicine, Keele University, Keele, UK. Electronic address: T.Gillingwater@ed.ac.uk.
authors.forename: Thomas H
authors.initials: TH
authors.lastname: Gillingwater
mesh_terms: D000818:Animals; D004195:Disease Models, Animal; D051379:Mice; D009134:Muscular Atrophy, Spinal / Q000378:metabolism / Q000473:pathology*; D055532:SMN Complex Proteins / Q000378:metabolism*; D013116:Spinal Cord / Q000378:metabolism*; D013997:Time Factors
chemical_list: D055532:SMN Complex Proteins
keywords: Biomarker; Clinical trials; Mouse; SMN; Spinal muscular atrophy; Western blot