18S rRNA, the Best Internal Control
A good internal control by definition has a constant expression level across the samples set being studied. Ambion recommends 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.
How to Use an Abundant Internal Control with a Rare Transcript
Ambion's 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).
Figure 1. QuantumRNA™ Technology in Multiplex Quantitative RT-PCR using 18S rRNA as an Internal Control. RT-PCR reactions on brain, embryo, liver, and spleen total RNA using A) primers for clathrin, B) primers for clathrin and 18S, or C) primers for clathrin,18S rRNA primers and 18S rRNA Competimers™. Note that without Competimers, 18S cannot be used as an internal control because of its high abundance (B). Addition of Competimers (C) makes multiplex PCR possible, providing sample-to-sample relative quantitation.
Gene Specific Primers that Work with 18S rRNA Primers
The research team at Ambion 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 Ambion's 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.
Figure 2. Multiplex Quantitative RT-PCR with Gene Specific Relative RT-PCR Kits. 1X (A) or 10X (B) amounts of the specific mRNA to be detected were added to replicates of a total RNA sample. The RNA was reverse transcribed with the RETROscript™ Kit and random decamers. Individual PCR reactions were performed with each reaction containing gene specific primers, 18S rRNA primers and 18S rRNA Competimers™. The patented Competimer technology attenuates 18S rRNA amplification efficiency so that it can be multiplexed effectively with the much less abundant interleukin targets.