Quantitation of Multiple RNA Species

Real-time quantitative polymerase chain reaction (PCR) methods (1 ) can be further optimized for various purposes by performing quantitative PCR for multiple RNA species in one sample (2 ). Advantages of this method not only include the elimination of differences in reaction mix volumes and conditions that may occur in separate samples, but importantly it also allows reference of RNA quantitation to an internal control (also see Notes 1 and 2 ). Internal controls are RNA species for which the quantity of RNA does not change across different cell types or conditions. They allow correction of RNA quantitation for different starting quantities of total RNA. Widely used examples of internal controls are 18S ribosomal RNA and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (3 ), and commercial standard kits for these are available. Different internal controls may be appropriate depending on the cell types or conditions being studied, and it is advisable to try several different controls when setting up a new assay. Quantitation of multiple RNA species is possible through the use of fuorescent probes, such as Taqman™ probes, as previously described.