TB #185: The Accuracy of Competitive RT-PCR: Using the Right Exogenous Standard

RT-PCR has revolutionized the analysis of RNA. Researchers are no longer restricted to characterizing RNA transcripts that are reasonably abundant or to detecting viral or bacterial targets in samples where their titers are high. RT-PCR makes it possible to accurately quantitate RNAs that are present at only a few hundred copies per sample. But RT-PCR must be carefully controlled to provide effective quantitation. Here, we detail an experiment designed to test the various types of exogenous standards routinely used in competitive RT-PCR. As expected, RNA competitors (especially those resistant to nuclease degradation) exhibited significantly greater accuracy and precision than DNA competitors and mimics.

Accurate quantitation of mRNA expression can be accomplished by RT-PCR using a competitive strategy. Competitive RT-PCR involves adding a known amount of PCR-amplifiable standard into an RNA sample and then amplifying the standard and target RNAs in the same reaction. The exogenous standard and the endogenous target use the same primers for amplification, thus a competition for amplification components including primers, dNTPs, and polymerase ensues. If the exogenous standard is designed to be amplified at the same rate as the target sequence, then the ratio of products obtained from the endogenous and exogenous targets at the end of the amplification reflects the initial ratio of target to standard. Since the amount of exogenous standard added to the RT-PCR is known, the amount of endogenous target in the RNA sample can be determined (multiply the concentration of the input standard by the ratio of target-specific PCR product to standard-specific PCR product). The key to successful competitive RT-PCR is the exogenous standard. The minimum requirements for a standard are: 1) that it share PCR primer binding sites with the endogenous target sequence, and 2) that the PCR product resulting from amplification of the standard be distinguishable from that of the target. The latter criteria is usually fulfilled by creating a small deletion in the exogenous standard. For accurate quantitation, 3) the exogenous standard must be amplified at the same rate as the endogenous mRNA target. Only then will it be possible to make meaningful comparisons of the amplified products to the initial concentrations of the PCR templates.

Competitor vs mimic
There are two basic methods for constructing an exogenous standard for competitive RT-PCR. The endogenous target can be cloned and modified to generate a construct referred to as a "competitor". Typically, the modification involves creating a deletion by PCR that is 10% of the endogenous target length (Figure 1). The competitor and endogenous target PCR products can then be separated by gel electrophoresis. The two bands are quantitated, providing the ratio of endogenous target to competitor. The second type of exogenous standard is called a "mimic." A mimic is a construct with PCR primer binding sites identical to the target molecule surrounding a DNA sequence unrelated to the endogenous target. Mimics are also usually designed to produce PCR products that are 10% larger or smaller than the endogenous target to allow effective separation and analysis by gel electrophoresis.

Figure 1. Competitor Design and Use in Competitive RT-PCR. Where two primers (P1 and P2) are being used for PCR amplification of a given target, two additional primers are used to generate a deletion construct for competitive RT-PCR. Primer P3 includes a T7 promoter primer at its 5Ė end, the P1 sequence in its middle, and a downstream target specific sequence at its 3Ė end. P3 creates the deletion and incorporates a transcription promoter for subsequent synthesis of an RNA competitor. P4 primes synthesis from the target cDNA at a site approximately 50 nucleotides downstream of the P2 binding site. The extra fifty nucleotides are essential for RNA competitors as it assures that the competitor is reverse transcribed as efficiently as the endogenous target. For instance, when random sequence primers are used for reverse transcription, the extra nucleotides are required for efficient priming and synthesis through the entire amplification region. When sequence specific primers are used, the extra sequence allows RNA structure to form around the primer binding site of the competitor as it would for the endogenous target.

RNA vs DNA
Once an RT-PCR standard construct has been produced, the researcher can either use the DNA standard directly in the RT-PCR experiments or convert the template to RNA by transcription. A DNA competitor or mimic can be added to the sample either before or after the mRNA is converted to cDNA. RNA competitors must be added to the sample mRNA prior to reverse transcription so that the sample and competitor are simultaneously converted to cDNA. Although using DNA competitors may be convenient, the failure of the DNA standards to control for RT efficiency can yield substantial error in Competitive RT-PCR.

An experiment was designed to test the relative accuracy of competitive RT-PCR using the various types of standards. A competitor template for cyclophilin was produced using the strategy shown in Figure 1. DNA and RNA competitors were synthesized by PCR and transcription, respectively. The two preparations were trace-radiolabeled, gel-purified, and quantified by comparing the incorporated radiolabel to the total radiolabel available in the synthesis reactions.

Two additional standards were synthesized. A second RNA competitor was transcribed from the cyclophilin competitor construct using Ambion's RT-PCR Competitor Construction Kit (no longer available). The RT-PCR Competitor Construction Kit transcription reaction incorporates modified nucleotides that render the resulting RNA transcript impervious to degradation by most nucleases, including RNase A. The fourth standard was a DNA mimic that was generated by doing extensive low-annealing temperature PCR cycles using lambda DNA and the cyclophilin specific PCR primers. A product of a reasonable size was gel-purified and quantitated by trace-labeling as described above.

Experimental design
Pilot RT-PCR experiments were performed to approximate the number of copies of the four exogenous standards required to amplify equivalent amounts of PCR product as the target cDNA. 100 ng of total mouse liver RNA and 1 femtomole of the RNA competitors were reverse transcribed. 0.5 µl samples of the total mouse liver RNA RT reaction was mixed with dilution series of the two DNA standards and the two competitor cDNAs. Gel electrophoresis and ethidium bromide staining were used for analysis. The initial experiments indicated that 100 ng of total mouse liver RNA, 5 attomoles of RNA competitor, 0.5 attomoles of DNA competitor, and 0.05 attomoles of DNA mimic generated approximately equal amounts of PCR product. The quantitative studies involved mixing the indicated amounts of standard and sample RNA, reverse transcribing the resulting samples, and PCR amplifying the target and standards templates using cyclophilin primers that had been radiolabeled using T4 polynucleotide kinase and [ gamma

³²P] ATP.

For each combination of mouse liver RNA and standard, duplicate reverse transcription reactions were performed. Four variations of the reverse transcription reactions were used to assess the effects of RT variability. MMLV-RT concentrations were varied between 5 and 10 U/µl and the amount of total mouse liver RNA in the four reactions ranged from 100-200 ng. One hour, 42°C incubations converted the RNA targets to amplification-competent cDNAs. PCR reactions were initiated by addition of 1 µl of each of the RT reactions. Thirty cycles were sufficient to amplify the targets to quantitatable levels. Gel electrophoresis was used to distinguish the target and competitor PCR products. Figure 3 shows a sample autoradiograph. Scintillation counting provided the relative ratios of target to competitor. The ratio was multiplied by the concentration of the input competitor to reveal the number of cyclophilin mRNAs present in 1 µg of total mouse liver RNA.

Quantitation results
The experiments incorporating the two RNA competitors, one with standard nucleotides and the second with modified nucleotides, revealed that 1 µg of total mouse liver RNA contained approximately 65ą7 attomoles of cyclophilin RNA (Figure 2). In contrast, the DNA competitor experiments indicated that only 6.0ą5.1 attomoles were present in 1 µg of total mouse liver RNA and the DNA mimic suggested that only 0.92ą0.67 attomoles were present (Figure 2).

DNA standards underestimate mRNA copy number
DNA competitors and mimics greatly underestimate the concentration of target molecules in a sample. The primary cause of this error is the efficiency with which individual RNA molecules are converted to amplification-competent cDNA. Experiments at Ambion indicate that only 5-20% of mRNA molecules are converted to cDNA. Of these conversion events, only a fraction actually result in cDNAs that include the entire region of mRNA that is to be amplified. Thus, when DNA competitors are used, researchers can expect to err by ten to one-hundred fold when quantitating their target of interest.

RNA standards are more precise than DNA standards
Perhaps the most important observation from the experimental data is that RNA competitors are far less affected by tube-to-tube variation than DNA standards. As illustrated in Figure 2, the standard deviation for RT-PCR experiments using RNA competitors is only about 10% of the mean value while DNA competitors and mimics vary by almost 100%. In fact, in the eight experiments with the DNA competitor, the calculated concentration for cyclophilin ranged from 1.1-15 attomoles per microgram of mouse liver (Figure 2), introducing greater than 500% error into the competitive RT-PCR experiment. The reason for this variability stems from the inherent inconsistency of the reverse transcription reaction. The efficiency of reverse transcription is highly dependent on the concentrations of the reverse transcriptase and sample RNA, the reaction conditions, and the reaction time. RNA competitors control for this variability because, like the endogenous target, they are dependent on the reverse transcription reaction to become amplification-competent cDNA. The DNA competitors, of course, are already able to support PCR amplification, and thus are cannot to control for tube-to-tube variability in the reverse transcription reactions.

DNA mimics fail to control for amplification efficiency
A third observation is the inaccuracy of the DNA mimic (Figure 2). In addition to having the problems of DNA, mimics are almost never amplify at rates equivalent to the target molecule. PCR efficiency depends not only on primer binding, but also on the sequence present between the two primer binding sites. Thus two sequences with little or nothing in common will invariably be amplified at different rates.

Standard vs. modified RNA competitors
The data support the use of RNA competitors in competitive RT-PCR. But RNA competitors are not infallible. RNA is susceptible to nuclease-induced degradation. Thus any nuclease that is introduced during competitor preparation or while removing aliquots for use in RT-PCR experiments will decrease the concentration of RNA competitor being used and thus will affect the accuracy of the data generated. In cases of nuclease contamination, the best case is actually one where the nuclease contamination is great enough to completely degrade the RNA competitor. This way, the researcher will not waste time or effort on additional RT-PCR reactions. The worse case is one where a slight contamination occurs. The data generated gets progressively less accurate as the concentration of the competitor decreases. Comparing data generated on different days will result in inaccurate conclusions.