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quantitative PCR was introduced in the beginning of the 1990s, several groups used internal synthetic standards in the reactions to account for variances in reaction efficiencies, but these are not mentioned, suggesting that they have fallen out of fashion. These standards were typically identical to the target but lacked a sequence in the middle to be able to distinguish the standard from the target. Most importantly, they had the same primer sequences as the target, indicating simple multiplexing of the standard and the target. These internal standards can be used to absolutely quantify mRNA targets (2) and could solve many of the problems identified in the article by Wong and Medrano. It is easy to imagine such a standard in real-time PCR where the two PCR products (standard and target) could be separated by probes emitting different colors. Synthetic RNA internal standards have three advantages. First, by spiking the RNA sample with known amounts of standards, efficiencies in the reverse transcription step is accounted for. Since the standard is converted into cDNA, differences between tubes in the efficiency of the PCR are accounted for as well. Second, there is no need to worry about which reference gene to use for normalization. Third, absolute quantification could easily be obtained by relating to a known amount of internal standard (2) instead of relating to a relative internal standard, as in the case of a housekeeping gene and an external standard curve. It is not difficult to understand why such synthetic RNA standards are not used today since it takes some effort to produce and handle them without degradation, but it is difficult to understand why they are not considered as a solution or a tool for mRNA quantification using real-time PCR.

Comment on Wong and Medrano's “Real-time PCR for mRNA quantification” BioTechniques 39:75-85 (July 2005)