Preclinical validation of a multiplex real-time assay to quantify SMN mRNA in patients with SMA

Abstract

Objective: To determine whether survival motor neuron (SMN) expression was stable over time. Methods: We developed a multiplex real-time reverse transcriptase (RT)-PCR assay to quantify SMN transcripts in preclinical blood samples from 42 patients with spinal muscular atrophy (SMA) drawn for three time points per patient; most blood samples were shipped to a centralized laboratory. Results: We obtained a sufficient amount (9.7 ± 5.6 μg) of good-quality total RNA, and RNAs were stable for up to a 3-year interval. This allowed RNA samples collected during a 9- to 12-month period to be analyzed in a single run, thus minimizing interexperimental variability. SMN expression was stable over time; intersample variability for baseline measures, collected during a 17-month interval, was less than 15% for 38 of 42 SMA patients analyzed. This variability was well below the 1.95-fold increase in full-length SMN (flSMN) transcripts detected in SMA fibroblasts treated with 10 mM valproic acid. Conclusion: Real-time quantification of SMN messenger RNA expression may be a biomarker that is amenable to multicenter SMA clinical trials.