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
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
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.