Fig. 2.

Validation of 18S rRNA and Competimers for normalization. The invariant expression of 18S ribosomal RNA makes it an ideal internal control for quantitative RNA analysis. However, because of its high level relative to the target mRNA, it is necessary to attenuate amplification of the 18S rRNA to the level anticipated from the target message. Top: to achieve this, with QuantumRNA Competimers, which are specially modified primers that cannot be extended, adjusting the ratio of functional 18S rRNA primers to Competimers modulates the efficiency of amplification of the 18S PCR product from the first-strand cDNA template. The linear range of 10–250 ng RNA as template was used for initial RT-PCR reactions with 18S rRNA specific primers (P) and 18S rRNA Competimers (C) and the 30-cycle amplification protocol described in materials and methods. Bottom: representative agarose gel of the PCR products obtained with a P-to-C ratio of 1:3 visualized by ethidium bromide staining and ultraviolet light. Predicted size of the 18S rRNA is shown at left; sizes of the DNA markers (M) are indicated at right. Lanes 1, 7, 13, no RT; lanes 2, 8, 14, cDNA template from RT-PCR using 250 ng RNA; lanes 3, 9, 15, 125 ng RNA; lanes 4, 10, 16, 75 ng RNA; lanes 5, 11, 17, 25 ng RNA; lanes 6, 12, 18, 10 ng RNA.