Proper data analysis is crucial to obtaining valid and relevant results from any experimental system, but is especially critical when assessing variations in mRNA expression of genes, the so-called transcriptome. Unlike DNA which is present in each cell throughout the life of the organism, RNA is transiently expressed and levels vary according to cell type, developmental stage, physiology and pathology. Therefore, quantification of RNA is context dependent and inherently variable. Extra vigilance must be exercised to ensure the data are technically accurate before they can be examined for biological relevance. It should always be remembered that mRNA expression does not necessarily correlate with protein expression and protein expression may not correlate with function. Regardless of the instrument used to perform quantitative real-time PCR, certain basic conditions should be met. The assay should be robust and fulfill all criteria for a good assay. A standard curve using a defined template should result in a slope, coefficient of determination (r2) and y-intercept that demonstrate good efficiency, accuracy and sensitivity. The baseline and threshold should be properly set. The amplification curves should demonstrate exponential amplification and be within the detection limits of the instrument. Proper controls should be included to monitor contamination and sensitivity of the assay. Genomic contamination should be assessed, if applicable. Data should be reported in a manner that allows the variability of results to be seen. Proper statistical analyses should be applied to the data.
What will be covered in this chapter? There are many applications utilizing real-time PCR, but the most common is the 5' exonuclease or TaqMan® assay used to quantify gene expression using a set of primers and a dual-labeled fluorescent probe. This chapter will focus on the preliminary analysis of data for this popular utilization of real-time PCR. All subsequent analyses will be based on the accuracy of the post-run preliminary analysis. Therefore, the samples in a real-time PCR run must be verified as valid before any further interpretation of the results is possible. Assay design, SYBR® Green I assays, normalization, relative quantification, along with more specialized uses and analyses will be covered in other chapters. Starting with the questions: are the primer/probes giving a good assay and what constitutes a good assay, standard curves, how to interpret them and use them to improve your assays, will be discussed. When you take the first look at the results of a real-time PCR run, what to look for and how to look will be described: how to set the baseline, the threshold, assess the controls and how to interpret the wide variety of amplification curves (or no curves) which might be encountered. Are the amplifications curves real? Are the no template controls (NTCs) negative? Examples are shown for specific instruments, but the principles should be applicable to all instruments. Illustrations of common problems that can arise will be described, so that they will be recognized if they occur. Data comparing the results obtained from nine different instruments using default instrument values or operator-adjusted values will be shown. An example of the basic principles of data reporting and statistics will be shown. This chapter should be especially helpful to researchers, such as core facility personnel, who have the opportunity to deal with the real-time PCR preliminary data analysis of numerous users.
A quick review of terminology used in preliminary data analysis is in order to promote understanding of why the analysis settings are so important. The Ct is defined as the cycle when sample fluorescence exceeds a chosen threshold above background fluorescence. This is also known as the Cp or crossing point. A positive Ct results from genuine amplification, but some Ct values are not due to genuine amplification and some genuine amplification does not result in a Ct value. Therefore, regardless of the terminology, the key word is 'chosen'. The Ct is determined by both the chosen baseline setting and the chosen threshold setting. These settings may be selected by the user or the user may accept the default values of the software. Values can be fixed or they can be adaptive. In most cases the reporter signal is normalized to a reference dye by dividing the raw fluorescence of the reporter by the fluorescence of the passive reference, to compensate for minor well-to-well variation. This value is referred to as the Rn, the normalized reporter signal. When the background value has been subtracted from the Rn, then this value is referred to as delta (A) Rn, the normalized fluorescence value corrected for the background.
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