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Real Time PCR mRNA Quantitation.
mRNA Quantitation by Real Time PCR
One of the great advantages of Real Time PCR is accurate mRNA quantitation. Historically, northern blotting and ribonuclease protection assays (RPAS) have been the gold standards in molecular biology research, since no amplification is involved in these methods. Some researchers have used in situ hybridization however, this is mainly a qualitative protocol rather than quantitative one. Quantitating gene expression by traditional methods such as northern blotting presents several problems. One of the main advantages of real time PCR in the quantitation and detection of mRNA is that running a Northern blot or PCR products on a gel is much more time-consuming. Techniques such as northern blotting and RPAs work very well, but require quite a lot of RNA, even more than is sometimes available. PCR methods are particularly valuable when amounts of RNA are low, since the fact that PCR involves an amplification step means that it is more sensitive. Traditional PCR Methods Don't Cut it for Quantitative Results Traditional PCR is only semi-quantitative. This is due to several factors, however one of these is due to the need to use ethidium bromide for staining. Ethidium bromide is insensitive, however there have recently been many new and more sensitive ways to detect PCR products. Another factor is that traditional PCR methods do not allow the observation of the amplification reaction reaction during the truly linear part of the amplification process, you only get to see the final result after many cycles of amplification. Running 20-40 cycles which is common in most PCR reactions, the amount of product reaches a plateau, which depends more on the amount of primers in the reaction mix than the amount of starting template in the sample which you need to quantify! There have been attempts to overcome these problems with the introduction of various competitive PCR protocols but they tend to be too difficult or cumbersome.
Real Time PCR to Quantify cDNA, mRNA and RNA Levels Real-time PCR was developed so that a higher accuracy result could be obtained from traditional PCR methods. The biggest advantage of real time PCR over traditional methods such as northern blotting and ribonuclease protection assays (RPAS) is that it uses much less starting material such as RNA or cDNA, and therefore it is much more suitable to precious samples such as clinical samples. Real-time PCR has several additional advantages over traditional PCR in that Real Time PCR is relatively easy to use, it is convenient also compared to some of these older methods (as long as one has access to a suitable real-time PCR machine). Disadvantages of Real Time PCR methods include higher costs, and a slightly higher learning curve for users to learn the software. Also real time PCR primer design is more difficult than traditional PCR primers. How Does Real Time PCR Quantify mRNA? Relative concentrations of DNA present during the exponential phase of the reaction are determined by plotting fluorescence of dye bound to DNA (for example SYBR green) against cycle number on a logarithmic scale (so an exponentially increasing quantity will give a straight line). A threshold for detection of fluorescence above background is determined. The cycle at which the fluorescence from a sample crosses the threshold is called the cycle threshold, Ct. Since the quantity of DNA doubles every cycle during the exponential phase, relative amounts of DNA can be calculated, e.g. a sample whose Ct is 3 cycles earlier than another's has 23 = 8 times more template. Amounts of RNA or DNA are then determined by comparing the results to a standard curve produced by RT-PCR of serial dilutions (e.g. undiluted, 1:4, 1:16, 1:64) of a known amount of RNA or DNA. As mentioned above, to accurately quantify gene expression, the measured amount of RNA from the gene of interest is divided by the amount of RNA from a housekeeping gene measured in the same sample to normalize for possible variation in the amount and quality of RNA between different samples. This normalization permits accurate comparison of expression of the gene of interest between different samples, provided that the expression of the reference (housekeeping) gene used in the normalization is very similar across all the samples. Choosing a reference gene fulfilling this criterion is therefore of high importance, and often challenging, because only very few genes show equal levels of expression across a range of different conditions or tissues. Real Time PCR mRNA Quantitation References Higuchi et al. (1992). "Simultaneous amplification and detection of specific DNA sequences." Biotechnology 10:413–417. Higuchi et al.. (1993). "Kinetic PCR: Real time monitoring of DNA amplification reactions." Biotechnology 11:1026–1030. Wawrik et al. (2002) Real-time PCR quantification of rbcL (ribulose-1,5-bisphosphate carboxylase/oxygenase) mRNA in diatoms and pelagophytes. Appl. Environ. Microbiol. 68:3771-3779.
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