A new reverse transcription-polymerase chain reaction method for accurate quantification
© Shiao; licensee BioMed Central Ltd. 2003
Received: 18 September 2003
Accepted: 09 December 2003
Published: 09 December 2003
Reverse transcription-polymerase chain reaction (RT-PCR) is a very sensitive technique to measure and to compare mRNA levels among samples. However, it is extremely difficult to maintain linearity across the entire procedure, especially at the step of PCR amplification. Specific genes have been used as baseline controls to be co-amplified with target genes to normalize the amplification efficiency, but development or selection of reliable controls itself has created a new challenge.
Here, we describe a new quantitative RT-PCR to compare two mRNA samples directly without the requirement of synthetic control DNAs for reference. First, chimeric RT primers carrying gene-specific and universal PCR priming sequences with or without a linker for size distinction were utilized to generate cDNAs. The size-different cDNAs were then combined in a single reaction for PCR amplification using the same primer set. The two amplified products were resolved and detected with gel electrophoresis and fluorescence imaging. Relative abundance of the two products was obtained after a baseline correction.
This methodology is simple and accurate as indicated by equal amplification efficiency throughout PCR cycling. It is also easily implemented for many existing protocols. In addition, parameters affecting RT linearity are characterized in this report.
The relative RT-PCR procedure first establishes a number of amplification cycles, which is within linear range, and then compares samples from separate reactions to procure the ratios . This procedure does not account for the variation among reactions in different PCR environments, such as specific contents of each tube and difference between wells of the thermal cycler. Although linearity is obtained, the slopes of lines, indicative of efficiency, may not be equivalent, as seen in Figure 1. Another approach is to co-amplify control DNAs with target genes in a procedure termed competitive RT-PCR [3, 4]. The diverse efficiencies among PCRs are normalized with a signal of the same control DNA co-amplified with every test sample. In order to assure that control DNA gives the same PCR efficiency as the target gene, the control needs to be constructed empirically to minimize variation during co-amplification. To accomplish this, a specific control is developed for each target gene. Because synthetic control(s) are often much more abundant than the target, PCRs of serial dilution of control(s) are carried out to determine accurately the relative quantity of target to control(s). These steps complicate the entire procedure and make the competitive RT-PCR cumbersome. Real-time RT-PCR, an automatic version of relative RT-PCR, provides an advantage in that it monitors product yield during amplification cycling [4–6]. The CT value, the minimum number of cycles beyond which the signal exceeds a predetermined threshold, is commonly used for quantification, with the assumption of approximately 100% or equal efficiency for all PCR in the early phases of amplification. Special algorithms are then applied to obtain the relative quantity. However, the legitimacy of such assumptions and mathematical models has been challenged by others [7, 8]. Although real-time PCR provides high-throughput quantification, it is still not accessible to everyone due to the high cost for the equipment, and it is not cost-effective for projects examining only a few sample pairs.
Constant ratio throughout PCR
Accurate ratio estimation
Linearity of quantification for known RNA ratios
Comparison among cell lines
To achieve accuracy of quantification for RNA transcripts requires the acquisition of reaction and detection linearity throughout the entire procedure. Since the first development of RT-PCR, numerous approaches have been formulated to improve the precision of protocols from RNA measurement, RT, PCR, signal detection and analysis, to final mathematical correction. In this study, we have explored many aspects of quantitative RT-PCR with emphases on the RT and PCR steps. Accuracy is enhanced by improving the linearity of the RT reactions and by eliminating variations among PCR reactions (exemplified in Figs. 4B and 5B for efficiency), as well as sample loading for gel electrophoresis since two samples were co-examined during these analyses. Although competitive RT-PCR also provides such advantages, quantification with the latter technique is complicated by the need of reliable controls and for accurate estimation. Also, these controls are usually purified RNA or DNA, which are much more abundant than the target sequences in the cell sample. Serial dilution of controls is required for competitive RT-PCR. The unique designs in the new technique, such as gene-specific RT primers, a linker of either 10 or 15 bases in the long RT primer, and co-purification of cDNAs generated from the short and long RT primers, are different from the Ambion IntraSpec kit and are apparently important innovations to obtain specific amplification and accurate quantification. The strengths of this new quantitative RT-PCR include: it is a direct comparison of two RNA samples with no need of synthetic control(s); it is simple with no complex issues arising from the use of synthetic control(s); and it is accurate. Although a slight variation of ratio estimation utilizing 1:1 volume ratio of long to short product from the same RNA for baseline correction is seen, this can be highly reduced by the use of 5:1 (Figs. 4 and 5) or 2:1 (Fig. 7) for correction. Up to 1 order of magnitude of the dynamic range has been accurately obtained using the new method. Further analyses for higher ratio differences are needed to establish the upper limit of dynamic range for this method.
The accuracy of this direct quantitative RT-PCR is also demonstrated in the comparison of the Collagen I expression among three cell lines (Fig. 7). Inclusion of 4 reactions for each pair is useful for cross validation of the corrected ratio. In general, two reactions, one for baseline correction and one of crude ratio between samples, are sufficient to determine the final ratio.
Although the new technique measures only relative amounts of RNA in the two samples, an absolute copy number can be calculated from a calibration curve of reference DNA with the same sequence and length loaded onto the same gel. Reference DNA can be generated by PCR and purified in large quantity, and the copy number is then estimated from the absorbance at 260 nm wavelength. The new technique can also be modified to use regular polyT as a RT primer to produce cDNAs for subsequent PCR of more than one gene. We have synthesized chimeric primers to generate first-strand short and long DNA from corresponding polyT-cDNAs. The two first-strand DNAs are combined and are analyzed as in the present protocol. Although the current version of this new methodology is not suitable for high throughput gene expression profiling, we are currently investigating the possibility of converting this direct quantitative RT-PCR into a real-time format. With this innovation, no multiple PCR with incremental cycles is needed to determine the minimal number of cycles. Signal acquisition, graphic plotting, and baseline correction can be streamlined by any real-time analyzer with sophisticated software.
A direct quantitative RT-PCR (Fig. 2) has been described to obtain accurately the ratio of gene expression among samples. Approaches to reduce analytical variation throughout the entire RT-PCR procedure have been also presented here in detail.
Trizol reagent was used to extract total RNA from a NRK-52E rat kidney cell line and its daughter lines , according to the manufacturer's direction with some modifications (Life Technologies, Gaithersburg, MD). In brief, cells were cultured in Dulbecco's modified Eagle's medium (Biofluids, Rockville, MD) supplemented with 4 mM L-glutamine, 5% calf serum, 100 unit/ml penicillin, and 100 μg/ml streptomycin, in 3 T-162 flasks until they reached confluence. After removal of the media, 15 ml Trizol reagent was added directly into the first flask and then transferred sequentially to the second and the third flasks. The 15 ml lysate was held for 15 minutes at room temperature before the addition of 3 ml chloroform and vigorous shaking for 15 seconds. Phase separation of organic and aqueous layers was accelerated by centrifugation at >12,500 × g for 15 minutes at 4°C. The upper aqueous phase was transferred into a new tube and 8 ml isopropanol was added, followed by centrifugation at 14,000 × g for 15 minutes to precipitate RNA. The pellet was dissolved in RNase-free water. The total RNA was quantified by a spectrophotometer (8452A, Hewlett Packard, Mississauga, Canada) at 260 nm and was then verified with a RiboGreen RNA quantitation kit following the manufacturer's instruction (Molecular Probes, Eugene, OR). The RiboGreen fluorescence binds efficiently to single-stranded nucleic acids without interference from proteins and remnants of RNA extraction reagents, if present, in RNA solution. Ribosomal RNA was provided in the kit for generation of a standard curve, which was then used for sample quantity estimation.
Chimeric primers for RT and primers for PCR.
Rat Collagen I
Rat Collagen I
The same as the VHL lower primer
An aliquots of the short and long cDNAs were combined directly for purification with a Microcon 50 size-fraction spin column (Millipore, Bedford, MA) to remove RT primers before PCR. A Platinum Taq polymerase kit for hot start PCR was used according to the manufacturer's instruction (Invitrogen). Briefly, PCR was carried out in a mixture of 20 mM Tris-HCl, pH 8.0, 50 mM KCl, 0.2 mM deoxynucleotides, 1.5 mM MgCl2, 0.2 μM of each primer, and 2 U Platinum Taq DNA polymerase. The PCR thermal control started at 94°C for 2 minutes initially to activate HotStart Platinum polymerase, followed by cycling of 94°C for 30 sec., 60°C for 30 sec., and 68 (or 72) °C for 1 minute for 25 to 40 times.
The amplified short and long products with 10 and 15 base-pair (bp) difference in length were resolved in 20% and 10% polyacrylamide gel (Invitrogen), respectively. The gel was then stained with SYBR Green I fluorescence (Molecular Probes). Fluorescence signal was detected and analyzed by a BioChemi imaging system equipped with LabWorks 4.0 software (UVP Inc., Upland, CA). Initial measurement using "area density" function, which requires manually bracketing product bands, did not give a consistent quantification from the same products. A function with computer-assisted band identification and integration of peak area capabilities was finally utilized to obtain fluorescence intensity and the ratio between short and long product from the same reaction. Log scale of fluorescence intensity was plotted against PCR cycle number to determine the amplification efficiency.
Quantification using a commercial comparative RT-PCR kit, IntraSpec, was performed according to the manufacturer's instruction (Ambion). The RT primers, Tag10 and Tag50, with 40-bp difference in length were provided and were designed to recognize the polyA tail of mRNA. RT reaction was carried out at 49°C for 2 hours. After purification of the Tag10 and Tag50 cDNAs by a DNA-binding spin column, the two cDNAs were combined in a single PCR using the lower primer provided in the kit and an upper primer, which was selected following the manufacturer's guidelines. The PCR was conducted as described above except the annealing temperature was reduced to 50°C.
The ratios of repeated experiments were expressed as mean ± standard deviation. The consistency of mean ratios among experiments with different permutations, such as PCR cycles and the choice of baseline correction, were tested by ANOVA using the StatView software (Version 4.5, Abacus Concepts, Berkeley, CA). Mean ratios were considered to be not different if the p value was higher than 0.05.
I would like to thank Dr. Lucy M. Anderson for her crucial comments and support, Dr. Ailian Zhao and Ms. Noureen Khan for discussion, and Dr. Robert Cheng for critical review of this manuscript.
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