Quantification of mRNA using real time RT-PCR the SYBR solution

Abstract

Commonly used methods to quantify RNA and DNA include Northern and Southern blotting, RNase protection assays and in situ hybridization. Although these methods are direct in that they analyze nonamplified RNA or DNA, they are of low sensitivity and require relatively large amounts of nucleic acid. Another method, thousands of times more sensitive than these traditional techniques, combines reverse transcription (RT) and the polymerase chain reaction (PCR). Although RT-PCR is an exquisitely sensitive and specific technique, obtaining quantitative data presents a difficult challenge (1-4). The goal of all quantitative PCR methods is to determine the initial number of molecules of a given target DNA from the amount of product generated during PCR. A major obstacle to achieving this goal is the exponential nature of PCR itself. Under ideal conditions, when the reaction efficiency is 100% (i.e., E = 1), the amount of product generated increases exponentially, doubling with each cycle of PCR. In practice, the efficiency of amplification may be considerably less than this and can vary substantially. Reaction efficiency depends on many factors, including the primer sequences, the length of the amplicon and its GC content, and sample impurities (5). These factors affect primer binding, the melting point of the target sequence and the processivity of the Taq polymerase. Importantly, amplification of exactly the same sequence in replicate tubes using the same PCR block can give substantial variations in efficiency values (e.g., E = 0.8-0.99) even when a master mix of reaction components is used (6). This occurs because of small sample-To-sample differences in cycling conditions, perhaps in different regions of the block (e.g., center versus edge), which lead to small variations in reaction efficiency. Because of the exponential nature of PCR, this results in substantial differences in product yield as the reaction progresses. A difference in efficiency of as little as 5% between two samples with the same initial copy number can result in one sample having twice as much product after 26 cycles of PCR (7). A further difficulty in obtaining quantitative data is that the relationship between the number of molecules of a target present in a sample at the start andat the end of the PCR reaction is linear only during the exponential phase of PCR. During PCR product accumulates exponentially initially, but then slows typically reaching a plateau. Various factors may contribute to this plateau effect, including the accumulation of polymerase inhibitors, the loss of Taq activity, and increasing reassociation of the sense and antisense strands of the amplicon (at the expense of primer binding) as the target product accumulates. Despite these difficulties, quantitative PCR methods have been devised that use equipment and techniques common to most molecular biology laboratories (a PCR block, agarose gel electrophoresis, and densitometry analysis). These methods endeavor to overcome the difficulties of tube-To-Tube variation in efficiency and the limitations imposed by the need for measurement during the exponential phase of PCR. These "end-point" methods separate the amplicon from other reaction components by agarose gel electrophoresis and quantitate by staining with ethidium bromide (EtBr) (8), incorporate radiolabeled nucleotides or primers followed by autoradiography or phosphoimaging, or use hybridization strategies such as Southern blotting using radiolabeled amplicon-specific probes, to measure the quantity of amplicon synthesized. However, to ensure that measurements are made during the exponential phase it is necessary to sample and analyze the product every cycle or to run multiple serial dilutions of each DNA amplified. Another approach is competitive RT-PCR, which has the important advantage that products can be analyzed when the PCR process has reached a plateau and not only during the exponential phase (9). In this method, an internal standard that shares the same primer sequences as the target is amplified in the same tube leading to competition for reagents. The internal standard product is designed to have a small difference in size to the target amplicon (or a unique restriction site) allowing the amplicon generated from the internal standard to be distinguished from the target amplicon by agarose gel electrophoresis and enabling both products to be quantified by gel densitometry. A series of PCRs are run with a fixed amount of cell or tissue cDNA and varying concentrations of competitor. A graph of the logarithm of the ratio of target amplicon intensity/competitor amplicon intensity versus concentration of competitor spiked into the reaction is linear. The concentration of target cDNA can be determined from this graph as the logarithm of the ratio of target amplicon intensity/competitor amplicon intensity is zero when the concentration of target and competitor were equal at the start of PCR (10-12). Competitive RT-PCR is an ingenious and elegant procedure that circumvents some of the substantial problems in making PCR quantitative. However, competitive PCR assays are labor-intensive, unsuited to high throughput requiring post-PCR processing for all reactions, have a limited dynamic range and are expensive to run. These substantial practical problems limit the utility of this technique for quantitating gene expression. © 2008 Humana Press.