Abstract Background: Measuring the allelic ratios of placental transcripts in maternal plasma permits noninvasive prenatal detection of chromosomal aneuploidy. Current methods, however, require highly specialized equipment (MALDI-TOF), limiting the widespread implementation of this powerful RNA single-nucleotide polymorphism (SNP) strategy in routine diagnostic settings. We adapted and applied the Transgenomic WAVE System and quencher extension (QEXT) for this purpose. Methods: The expressed SNP (rs2187247) in exon 2 of the placentally expressed C21orf105 gene (chromosome 21 open reading frame 105) on chromosome 21 was tested in a trisomy 21 model system in which we obtained RNA selectively released from syncytiotrophoblasts of normal and trisomy 21 placentas during first trimester. Results: In identifying heterozygous samples, we observed an exact correspondence between sequencing results and results obtained with the WAVE System. With respect to the analysis time required, the WAVE System was superior. In addition, the real-time QEXT assay (as optimized and validated with calibration standards consisting of 262-bp C21orf105 cDNA amplicons) accurately measured allele ratios after we optimized fragment purification, concentrations of input DNA and quencher label, and calculations of reporter signals. Finally, the optimized and validated QEXT assay correctly distinguished normal placentas from trisomy 21 placentas in tests of the following clinically relevant combinations: diploid homozygous (CC), diploid heterozygous (AC), triploid homozygous (AAA), and triploid heterozygous (AAC or ACC). Conclusion: The QEXT method, which is directly adaptable to current real-time PCR equipment, along with rapid identification of informative samples with the WAVE System, may facilitate routine implementation of the RNA-SNP assay for noninvasive aneuploidy diagnostics.