A good internal control by definition has a constant expression level across the samples set being studied. We recommend using 18S rRNA as an internal control in relative RT-PCR because it shows less variance in expression across a variety of treatment conditions than β-actin and GAPDH. However, because 18S rRNA is so abundant, it amplifies rapidly during RT-PCR, quickly exhausting the reaction reagents. It can, therefore, be difficult or even impossible to detect product from rare messages while remaining in the exponential phase of amplification for 18S rRNA.
Our patented Competimer™ Technology provides a way to use an abundant internal control like 18S rRNA in reactions where a rare message is being coamplified. The Competimer Technology allows 18S rRNA amplification to be attentuated to the level of rare messages. Attenuation results from the use of competimers-primers identical in sequence to the functional 18S rRNA primers but that are "blocked" at their 3'-end and, thus, cannot be extended by PCR. Competimers and primers are mixed at various ratios to reduce the amount of PCR product generated from 18S rRNA. Figure 1 illustrates that 18S rRNA primers without competimers cannot be used as an internal control because the 18S rRNA amplification overwhelms that of clathrin (Figure 1, Panels A, B). Mixing primers with competimers at a 3:7 ratio attenuates the 18S rRNA signal, making18S rRNA a practical internal control (Panel C).
Our research team has also generated over 100 Gene Specific Relative RT-PCR Kits. These kits contain primer pairs for specific human, mouse and rat genes, positive control DNA, a detailed Instruction Manual, and our exclusive QuantumRNA 18S rRNA primers and competimers. Kits are available for analysis of apoptosis genes, cytokines, cytokine receptors, growth factors, growth factor receptors and oncogenes in human, rat, and mouse. Typical data generated via gene specific relative RT-PCR is shown in Figure 2.