Applied Biosystems 7900HT Fast Real-Time PCR System and SDS ...

Chapter 6Analyzing Real-Time DataEssential Experimental ComponentsEndogenousControlCalibratorSampleAll relative quantification experiments require data from a second probe and primerset that amplifies an endogenous control sequence. Endogenous control targets aretypically constitutive RNA or DNA sequences that are present at a statisticallyconsistent level in all experimental samples. By using the endogenous control as anactive reference, the data from the amplification of a messenger RNA (mRNA) targetcan be normalized for differences in the amount of total RNA added to each reaction.Examples of common endogenous controls are: GAPDH, 18S rRNA, and β-Actin.The endogenous control can be amplified independently of the target sequences inseparate wells on the reaction plate (as singleplex or non-multiplex reactions), or inthe same well (as multiplex reactions).IMPORTANT! For non-multiplex experiments, the reactions amplifying theendogenous control must be located on the same plate as the target assays.IMPORTANT! To generate a standard deviation for the relative quantity value of atarget, each plate must contain usable data from at least two replicates of the targetand endogenous control.All relative quantification experiments require data from a calibrator sample. Duringanalysis, the SDS software calculates gene expression levels in samples relative tothe level of expression in a calibrator sample. Thus, the calibrator plays an integralpart in the calculation because it is used as the basis for the comparative results.Examples of possible calibrator samples include:• A zero time-point sample in a time-course experiment• An untreated sample (versus treated samples)• A resting sample (versus activated samples)Note: The SDS software combines the data from replicate calibrator wells at the ∆C Tlevel of the relative quantification calculation (whether the replicates are present on asingle or multiple plates).Replicate WellsFor relative quantification studies, Applied Biosystems recommends the use of threeor more replicate reactions per sample to ensure statistical confidence.Replicates allow you to:• Preserve Data – If an amplification fails in one well, replicate wells canpotentially provide data. This point is especially true in the case of endogenouscontrols which, upon failure, may invalidate the results for the entire plate.• Remove Outliers – The use of replicate populations provide the opportunity forthe visualization and removal of outliers.• Ensure Statistical Reproducibility – In general, the use of replicates ensures agreater degree of experimental reproducibility by providing a means to identifyand refute anomalous data caused by experimental error (such as contamination,pipetting errors, and so on).6-20 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Real-Time.fm