Qualitative and Quantitative DNA and RNA Analysis by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry - Clinical Applications of PCR - page 64

Fig. 2. Schematic representation of allele-specific quantification. When both the
wild-type and the mutation DNA are present, the competitor is designed to have a
synthetic mutation next to the natural mutation. Three extension products from the
wild-type DNA, the mutant DNA, and the competitor are designed to have different
molecular weights, and thus can be detected by matrix-assisted laser desorption/ion-
ization time-of-flight mass spectrometry.
designed such that the molecular weight values of the aforementioned four prod-
ucts ( Fig. 2 ) differ by at least 25 Da from each other.
3.1.3. rcPCR for cDNA Quantification and cDNA Mutation Detection
and Quantification
Generally, assay designs for cDNA quantification (or gene expression analy-
sis) are very similar to DNA quantifications. To avoid potential genomic DNA
contamination, it is better to design a PCR amplicon spanning a long intron. If
this is not possible, a negative control experiment using nonreverse-transcribed
RNA samples should be performed.