A Practical Approach, Second Edition=Ronald D. Ho.pdf

610 DEVELOPMENTAL REPRODUCTIVE TOXICOLOGY: A PRACTICAL APPROACH, SECOND EDITION2600Florescence24002200200018001600140012001000800600400200Threshold0Threshold Cycle353025y = 44.877 − 3.4109x R 2 = 0.999203.0 4.0 5.0 6.0 7.0Log Concentration (molecules)10 7.0 molecules10 6.0 molecules10 5.5 molecules10 5.0 molecules10 4.5 molecules10 4.0 molecules10 3.5 moleculesNegative Control−20010 20Cycle Number30 40Figure 14.3The plot illustrates a typical real-time RT-PCR experimental run in which the increase in fluorescenceis tracked by cycle number. The samples are DNA standards with 10 7 to 10 3.5 moleculesper reaction tube at ½ log intervals, except for the 10 6.5 interval, which was not run. The insetgraph is a plot of the initial concentration of DNA vs. the threshold cycle, the cycle at which eachtube exhibited the same level of amplification.hydrolysis probes, these are also end-labeled with a reporter and quencher. However, the ends ofthe probe contain short complementary sequences designed to allow the probe to form a stem-loopstructure when free in solution. This results in the reporter and quencher being in very closeproximity and having low background fluorescence when compared with hydrolysis probes. Whenthe probe hybridizes to the specific amplicon, the stem-loop structure opens, the distance betweenthe reporter and quencher increases, and the reporter fluoresces. As the PCR primers are extended,the probe is displaced rather than being degraded.Figure 14.3 illustrates a typical real-time RT-PCR experiment that measured the increase influorescence tracked by cycle number. The curve for each sample is characterized by a region whereinsufficient product has been synthesized to allow detection, followed by an exponential increasein the accumulation of product, and finally by the appearance of a plateau. The relative amount ofmRNA in each sample is determined by comparing the cycle number when the fluorescence reachesa threshold level. The threshold level is selected at a point sufficiently above the noise level of theinstrument to obtain accurate fluorescence measurements while amplification is still occurringexponentially. The amount of mRNA in the original sample is inversely proportional to the thresholdcycle number. Reproducibility between real-time RT-PCR runs is sufficient that for many purposesthe use of standards is not required. 47 Standards can be used to reduce run-to-run variability. Thesestandards can be either cDNA or RNA samples. If it is necessary to determine the actual numberof copies of a specific mRNA contained in a given sample, it is important to control for the efficiencyof the reverse transcription reaction. This can be accomplished by including a standard curve derivedfrom a pure RNA sample of the sequence being amplified with each run.© 2006 by Taylor & Francis Group, LLC