Once the initial examination and analysis of the preliminary data have been completed, the next step is to decide how to compare the data in a meaningful manner. Figure 2.11 illustrates the general concepts of secondary analysis. Normalization and relative quantification are extensive topics that are covered in other chapters. Using a standard curve for quantifying mRNA or DNA is sometimes referred to as absolute quantification, but the term absolute is debatable since it is only as accurate as the measurement of the material used for the standard curve. This method is better
Secondary analysis flowchart
'Absolute' quantification using an external standard
Relative quantification compared with other gene(s)
Adjusted for efficiency REST, Q-Gene, DART
Not adjusted for efficiency
Graphical, tabular representation
Flow chart for secondary analysis. Suggested steps for analysis of real-time PCR data after the preliminary data has been verified.
referred to as quantification using a standard curve. The standard curve allows the amount of unknown samples to be computed on a per cell or unit mass basis. But, no matter how accurately the concentration of the standard material has been determined, the final result is relative to a defined unit of interest (Pfaffl, 2004).
The characteristics of a standard curve are shown in Figure 2.1 and described in the section on standard curves. Most real-time instruments have software that will calculate the amount of unknown values in the same units designated in the standard curve, but unknowns can be calculated in a spreadsheet using the formula:
Log10 copy number = Ct - y-intercept/slope
It is important to remember that verifying your reagents are capable of giving a 100% efficient reaction, as described in the section on standard curves earlier in this chapter, does not necessarily mean that particular template will give an accurate measure of gene expression in your sample. Using a defined template, such as a plasmid, PCR product or synthetic oligonucleotide, while easy to prepare, does not take into account the sample preparation and reverse transcription step. Transcribed RNA can be used to control for the reverse transcription step, but it does not control for template preparation and is quite labor intensive. Genomic DNA can be used for the standard curve and may be a more realistic control for sample preparation, but is only useful if the primer/probe set are designed within one exon. Obviously, there is not one perfect method for quantification. Be aware of the pitfalls and choose the method which is best suited to your goals.
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