Semi-competitive RT-PCR was developed to quantitate values of target RNA. The procedure relies on the use of an external standard that mimics or closely imitates the target RNA species with respect to primer binding and other variables affecting PCR amplification. This is an important difference from the exogenous internal standard used in the noncompetitive RT-PCR, where the standard primer set can amplify a completely different target than the experimental target mRNA. In the competitive assay, the standard is similar enough to the target that competition with target occurs and, ideally, the experimental and standard target sequences amplify with the same efficiency but can be distinguished from each other following agarose gel electrophoresis.
These standard fragments fall into two categories: homologous and heterologous competitor fragments.
Homologous fragments differ only slightly from the target sequence with the addition of a unique restriction site or the presence of an additional sequence such as an intron that increases the molecular weight of the standard. One potential problem with this sort of standard is the formation of heteroduplexes between the standard and target sequences during the amplification process-an artifact that could interfere with quantitation.
Heterologous competitor fragments differ from the target except for the flanking primer-template regions, which are identical. Thus, heteroduplex formation cannot occur and slight differences in the target and primer sequence can be easily created.
A major source of variability usually not controlled for in competitive RT-PCR assays involves RNA purity and RNA integrity. In assays where DNA standards instead of RNA standards are used, there is also no control for reverse transcriptase yield and uniformity. Because the nature of the RNA preparation is a major source of variation in any assay of gene expression, the reliability of this approach without additional endogenous internal standards is suspect. However, because most investigators are interested in quantitating relative differences between control and treatment groups, the necessity for exogenous mimic RNA standards to attempt to determine absolute levels may be limited.