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

From Sainte-Justine Hospital Research Centre (L.R.S., M.-C.B., S.M.), Montreal, Canada; University of Utah School of Medicine (M.W., K.J.S.), Salt Lake City; and Department of Pathology (T.W.P.), The Ohio State University, Columbus.

Address correspondence and reprint requests to Dr. Louise R. Simard, Professor & Head, Biochemistry and Medical Genetics, University of Manitoba, Faculty of Medicine, 770 Bannatyne Ave., Winnipeg, Manitoba R3E 0W3, Canada; e-mail: louise_simard{at}umanitoba.ca

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.

Additional material related to this article can be found on the Neurology Web site. Go to www.neurology.org and scroll down the Table of Contents for the February 6 issue to find the title link for this article.

This work was funded by Families of SMA. Additional funding was provided by the Muscular Dystrophy America (KJS) and by the Spinal Muscular Atrophy and American Academy of Neurology Foundations (Young Investigator Award, KJS).

Disclosure: The authors report no conflict of interest.

Received March 2, 2006. Accepted in final form October 20, 2006.

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