Different therapeutic strategies are currently evaluated in spinal muscular atrophy (SMA) that are aimed at increasing full-length (FL) mRNA levels produced from the SMN2 gene. Assays measuring SMN mRNA levels are needed. We have developed a sensitive, comparative assay based on multiplex fluorescent reverse-transcription polymerase chain reaction (RT-PCR) that can measure, in the same reaction, the levels of SMN mRNA with and without exon 7 sequences as well as those of total SMN mRNA. This assay allows to calculate directly the ratios of FL SMN mRNA to SMN mRNA without exon 7 (Delta7). We have used this assay to compare the levels of SMN transcripts in the blood of 75 unrelated normal subjects and of 48 SMA patients, and in muscle samples of 8 SMA patients. The SMN1 and the SMN2 genes produced very similar levels of total mRNA. Levels of transcripts lacking exon 7 were linearly dependent on the number of SMN2 copies, both in SMA patients and in controls. In patients, FL mRNA levels correlated with SMN2 copy number. A significant but weaker inverse correlation was also observed between FL or Delta7 mRNA levels and disease severity, but patients with three SMN2 copies and different SMA types displayed similar mRNA levels. A significantly higher FL to Delta7 ratio was measured in blood cells than in skeletal muscle (0.80+/-0.18 versus 0.47+/-0.11). This assay can be used as a sensitive biomarker for monitoring the effects of various drugs in forthcoming clinical trials of SMA.