荧光定量原理与应用(第二版).pptVIP

* Comparative Ct methods can be broken down into two categories. Normalized Expression and Relative Quantity Relative quantity is calculated by the equation E^delta Ct where delta Ct is the difference between a chosen sample and any given sample in the experiment. This information is available in the iQ5 online help as well as some documentation supplied in training. The value is a non normalized value. No correction for loading or cell number is present in the equation. The researcher typically takes great care to ensure s/he is loading exactly the same amount of nucleic acid for each and every sample tested. Normalized expression as implied by the Name is an “adjusted” value. This equation does account for loading or cell number differences assuming acceptable reference genes are chosen. Normalized expression is simply the Relative Quantity of the gene of interest divided by the Relative Quantity of the reference gene. If more than one reference gene is used then the geometric mean of the relative quantity of all reference genes is the denominator. * This maps out the “features” of the popular methods used to evaluate normalized expression. Note that delta delta Ct is the simplest approach. It assumes perfect PCR reactions for all assays and can only account for one reference gene. The equations presented by Vandesompele can account for differences in PCR efficiency and can normalize to multiple reference genes. Teaching these as separate approaches is convenient but it is important to keep in mind that they are all variations of the same mathematical models. This is easiest to see when comparing Pfaffl’s approach to Vandesompele’s. The former is the same as the later except that it has a simpler denominator. You must remember your rules of exponents, set E = 2 and rearrange the Pfaffl equation in order to get the equation we normally think of as Livak’s delta delta Ct equation. * It’s important to note that some customers run a reference (such as