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


CONCLUSIONS
Real-time quantification of SMN messenger RNA expression may be a biomarker that is amenable to multicenter SMA clinical trials.
Simard
L R
LR
Sainte-Justine Hospital Research Centre, Montreal, Canada. louise_simard@umanitoba.ca
Bélanger
M-C
MC
Morissette
S
S
Wride
M
M
Prior
T W
TW
Swoboda
K J
KJ
D015415:Biomarkers; D015331:Cohort Studies; D003199:Computer Systems; D017362:Cyclic AMP Response Element-Binding Protein / Q000032:analysis* / Q000235:genetics*; D020022:Genetic Predisposition to Disease / Q000235:genetics; D006801:Humans; D009134:Muscular Atrophy, Spinal / Q000175:diagnosis* / Q000235:genetics*; D009419:Nerve Tissue Proteins / Q000032:analysis* / Q000235:genetics*; D010865:Pilot Projects; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics*; D016601:RNA-Binding Proteins / Q000032:analysis* / Q000235:genetics*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D055532:SMN Complex Proteins; D012680:Sensitivity and Specificity
D015415:Biomarkers; D017362:Cyclic AMP Response Element-Binding Protein; D009419:Nerve Tissue Proteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D055532:SMN Complex Proteins