Basically, two applications of RT-PCR are the most common.

1. RACE: cloning of 5' ends using a gene-specific 3' primer during reverse transcription, and *some* method to anchor a 5' primer to provide the upstream priming site for subsequent PCR.

2. Quantitative PCR: a method to measure very small amounts of RNA transcripts as cDNA targets. There are two strategies currently used, one is called Competitive RT-PCR and the other (more advanced) is called Real-Time PCR.

RACE: Rapid Amplification of cDNA Ends

The RACE RT-PCR strategy was first described in 1988 by Michael Frohman and Gail Martin. The original protocol used a gene specific primer and polyadenylation of the extended cDNA at the 3’ end with terminal deoxytidyl transferase (TdT).

The Frohman and Martin RACE protocol utilizes a TdT-mediated polyadenylation reaction to create the upstream "anchored" cDNA priming site for PCR amplification. To increase specificity during PCR amplification, a "nested" gene-specific primer (GSP2) can be used in conjunction with an anchor primer lacking the oligo dT sequence (AP). Subcloning of the RACE product is facilitated by including restriction site sequences in the GSP and AP primers.

Several modifications of the original RACE protocol have been described. One of these is called RNA ligase-mediated RACE (RLM-RACE), and it involves the use of bacteriophage T4 RNA ligase to covalently attach a single strand RNA anchor molecule to the de-capped 5’ end of mRNA. First strand cDNA synthesis can then be performed using a gene-specific primer to produce a pool of cDNAs encoding the anchored primer sequence.

The RLM-RACE strategy was developed by Ambion. The basic idea is that RNA ligase will only ligate on the anchor RNA sequence to RNA molecules that contain a 5' Cap structure. The problem with this strategy is the inefficiency of RNA ligation of two single strand molecules.

What could be a problem with using RACE to clone the 5' end of a transcript that contains the amino terminal coding sequence of a novel protein?

What could you do to minimize this problem? How might you be able to confirm that the RACE product represents the bona fide gene product you are interested in?

If at first you don't succeed, and you want to try, try again, what five parameters would you vary in your PCR reaction conditions if you are not generating the desired product?

What cloning "problem" might you encounter in conventional cDNA library construction that could possibly be overcome by RACE? Explain.

Quantitative RT-PCR Strategies

It is often important to know relative steady-state levels of specific gene transcripts as a means to investigate the role of a gene function in a particular cell phenotype. Two common ways to measure steady-state mRNA levels, Northern blots and RNase protection assays, are labor-intensive and may require up to 25 micrograms of total RNA for each assay.

Meaningful quantitative RT-PCR measurements require a standard RNA template against which the experimental RNA can be measured. Two types of RNA standards have been used for this purpose. One type is an endogenous gene product that is ubiquitously expressed in all cell types, for example, actin or glyceraldehyde 3-phophate dehydrogenase (GAPDH).

The other type of standard is an exongenously added cRNA (complementary RNA) that is synthesized by in vitro transcription. These exogenous cRNAs are sometimes called RT-PCR "mimics" because they contain the same RT-PCR priming sites and overall sequence as the target RNA, but produce a PCR product that differs from the target RNA by a unique restriction site or shift in molecular weight. Mimic cRNAs are transcribed from plasmids containing SP6 or T7 bacteriophage promoters and are constructed by in vitro mutagenesis.

Shown below is a flow scheme illustrating how insertional mutagenesis can be used to construct an AMG mimic cRNA for use as an internal marker in RT-PCR reactions using AMG gene-specific primers (P1 and P2). The AMG mimic PCR product is 30 bp longer than the normal AMG PCR product.

Competitive RT-PCR is done by performing a series of RT-PCR reactions that contain the same amount of sample RNA to which various known amounts of mimic cRNA have been added. The concentration of mimic cRNA that produces the same amount of mimic PCR product as the target RNA in the sample (a mimic:target product ratio of 1.0), represents the point at which both templates are competing equally for the primers.

Since competitive RT-PCR quantitation is based on ratios, it is not necessary to restrict PCR amplification to the exponential phase, and therefore, maximum sensitivity can be achieved by performing up to 35 cycles. A plot of log10 ratio of AMG mimic product/AMG target product versus log10 amount of AMG mimic cRNA added to the RT-PCR reaction expressed as molecules/cell can be used to calculate the amount of AMG target in the sample. This is equivalent to the reaction condition in which both the AMG mimic and target templates are competing equally for AMG primers

Quantitative Real-Time PCR
This technique is based on the use of a fluorescent oligonucleotide detection system in which the amount of fluorescence is proportional to the amount of PCR product in the reaction after each cycle. By using a standard curve based on known amounts of target DNA, it is possible to accurately and reproducibly quantitate the amount of target DNA in a test sample.

The principal of Real-Time PCR is illustrated below. This example uses a TaqMan oligonucleotide strategy from Applied Biosystems. The single strand oligo probe is designed to anneal to sequences within the PCR product and contains two fluorochromes. The 5' fluorochrome is called the "reporter" (R) and is usually 6-carboxyfluorescein (6-FAM), whereas the 3' fluorochrome (6-carboxy-tetramethyl-rhodamine; TAMRA) functions as a Quencher (Q). Multiple reporter fluorochromes are available to permit multi-plexing PCR reactions for controls.

The probe is designed to have a higher Tm than the primers, and during the extension phase, the probe must be 100% hybridized to the PCR product for the assay to be accurate. As long as both fluorochromes are present on the same molecule, no fluorescence is observed. However, once the Taq DNA polymerase initiates the next round of synthesis, the inherent 5'-3' exonuclease activity of the enzyme degrades the TaqMan probe and releases the Reporter fluorochrome from the quenced state. Fluorescence is recorded on a continuous basis and the PCR amplifcation process is monitored throughout the reaction.

Real-Time Quantitative RT-PCR is easy to do and more reproducible. The primary advantage is that the fluorescence detection system provides a large dynamic range which permits high throughput sampling independent of target number variability in paired samples. The method permits routine identification of 10 to 10,000,000 targets per sample.

Standard PCR using test primers and known amounts of target DNA (35 cycles of amplification).

TaqMan probe used in Real Time PCR sample amplification

CT = cycle at which the observed fluorescence is 10-fold above background (10x amplification).

Generating a standard curve using known amounts of target DNA and measuring the CT for each reaction.

Using the standard curve above, what is the estimated target DNA copy number in the sample amplification?

Do you think that it would be necessary to include internal controls in Real-Time PCR assays, such as with actin or GAPDH primers? Explain how this could be done in a single reaction tube.

Why is it necessary to test the amplification reaction conditions using gel electrophoresis before collecting data with the GeneAmp Real-Time PCR assay?