Validation of competitive RT-PCR strategy by use of a nonhomologous DNA fragment (RET_Comp ) engineered to contain specific RET primer templates. (A) Kinetics of amplification of RET cDNA and RET_Comp fragments. Fixed amounts (0.1 attomoles) of RET cDNA and RET_Comp fragments were amplified in a single reaction tube with specific RET primers. After 22 amplification cycles and after each of 8 additional cycles, a small aliquot of the reaction mixture was removed and the products were resolved on agarose gel (upper panel). The relative intensities of the bands corresponding to RET cDNA (790 bp) and RET_Comp (597 bp) -amplified products were quantified by computer imaging. The amount of specific amplified products for RET cDNA (○) and RET_Comp (•), expressed in AU, was plotted as a function of the number of cycles (lower panel). (B) Determination of relative levels of RET mRNA in THP-1 cells by competitive RT-PCR. Ten-fold serial dilutions (10 to 1 × 10⁻⁵ attomoles) of RET_Comp were amplified with RET primers together with constant aliquots of cDNA from the THP-1 cell line. After 35 cycles, amplified products were resolved on agarose gel (upper panel). Relative intensities of the bands were densitometrically determined and the logarithm of their ratios was plotted as a function of the logarithm of the amount of RET_Comp added (lower panel). The equivalence point (arrow) was inferred between 10⁻¹ and 10⁻² attomoles.