Initially, nucleic acid quantification meant the addition of radiolabeled UTP or deoxythymidine triphosphate (dTTP) to cell cultures or one of many possible in vitro experimental preparations and measuring their incorporation into nucleic acids by TCA (trichoroacetic acid) precipitation. Although radioactive incorporation is a quantitative technique and gave the investigator an idea of the global changes in the nucleic acid population of their experimental system, it was not satisfactory for identifying or quantifying specific genes or transcripts. The first breakthrough in the identification of specific genes came with the development of the Southern transfer method (Southern, 1975). This was followed by the Northern blot for RNA (Alwine et al., 1977). In both cases, it was now possible to specifically identify a particular gene or transcript by hybridization of a radioactively labeled probe to a membrane bearing DNA restriction fragments or RNA. Neither of these methods were quantitative techniques despite the best efforts to extract quantitative information from their use.
The next improvement in RNA transcript quantification came with the RNase-protection experiment. In this method, a short (<500 bases) highly radioactive anti-sense RNA was made from a plasmid construct utilizing T7 RNA polymerase. In vitro transcribed RNA was synthesized for the transcript(s) of interest along with a probe used for loading normalization. The radioactive anti-sense probes were then combined with each total RNA sample and hybridized to completion in liquid. The resulting double-stranded RNA product(s) were protected from the subsequent addition of a cocktail of single strand-specific nucleases. The protected products were then separated on a gel via denaturing polyacrylamide gel electrophoresis (PAGE) and exposed to film. This method had the advantage of hybridization in liquid rather than a solid surface and did not require the transfer of the RNAs to a membrane. However, the inherent short dynamic range when using film was the same. Again, the phosphoimager improved greatly on quantification for these experiments due to the expanded dynamic range and improved software for analysis. The down side to RNase protection experiments was the probes had to have high radioactive specific activity and required great care for safety reasons.
The procedure for performing the polymerase chain reaction (PCR) was first introduced by Kerry Mullis in 1983 (Mullis, 1990) for which he won the Nobel Prize in 1993. It is hard to think of another laboratory technique that has had a greater impact on so many different facets of biological research than PCR. In combining the reverse transcriptase (RT) reaction with the PCR, identification of a specific RNA transcript was now possible from very low copy numbers of starting material. Quantification of transcripts from sample unknowns became possible with the advent of competitive RT-PCR (Vu et al., 2000). In this method, a truncated version of the target region of interest lies between the same primer-binding sites as the target transcript sequence within a plasmid clone. The easiest method for making a smaller competitive target was to digest the cloned region between the primerbinding sites with a restriction enzyme and then ligate the resulting sticky ends, dropping out a short section of sequence. The requirements for the quantification construct were that it be a similar, but different, size than the target PCR product and quantified. The plasmid contains a T7, SP6 or T3 RNA promoter sequence up stream of the cloned target sequence. Utilizing the RNA promoter, truncated in vitro transcribed RNA could be made and quantified. Known amounts of the RNA product were spiked into the RT reaction and converted into cDNA along with the target sequence within the unknown sample. Subsequently, both the truncated standard and unknown target sequences were amplified using the PCR. The amplified DNAs were separated using denaturing polyacrylamide gel electrophoresis. In some methods a radioactive deoxynucleotide base was added for labeling the amplified DNA and quantified using either film or a phosphoimager. In other methods, the products on the gel were imaged following staining with ethidium bromide or SYBR® Green I. Quantification of the unknown target band was determined by comparison to signal from the spiked and quantified DNA standard. Although this method was the most accurate to date it still suffered from the detection problems mentioned earlier. However, most of the criticism centered around the spiked DNA standard. The concern was that the DNA standard was competing for reagent resources and primers with the unknown target during the PCR and might, therefore, alter the final result.
In 1996, Applied Biosystems (ABI) made real-time PCR commercially available (Heid et al., 1996) with the introduction of the 7700 instrument. Real-time quantitative PCR has become the most accurate and sensitive method for the detection and quantification of nucleic acids yet devised. This method has overcome most of the major shortcomings of the preceding ones. Using the specificity and sensitivity of PCR combined with direct detection of the target of choice utilizing fluorescently labeled primers, probes or dyes, the inherent problems of gels, transfer to a membrane, radioactive probe hybridization and the limitations of film as a detector have been eliminated. There are two problems for real-time PCR that still linger. They are 1) methods of quantification, i.e. kind of standard, assay quality and calculation methodology used and 2) how to properly normalize different samples to correct for differences in nucleic acid input from sample to sample. Both of these areas are the topic of much study and will be discussed in more detail in following chapters.
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