Technical Review Real time PCR
Normal PCR: Analyze with agarose gel
Normal PCR vs Real time PCR
Real-time PCR, also known as quantitative PCR (qpcr) or kinetic PCR Key feature: Used to amplify and simultaneously quantify a targeted DNA or cdna molecule in real time. Enables both detection and quantification (as absolute number of copies or relative amount when normalized to DNA input or additional normalizing genes) of a specific sequence in a DNA sample.
Frequently, real-time PCR is combined with Reverse Transcription PCR to quantify low abundance mrna, enabling a researcher to quantify relative gene expression at a particular time, or in a particular cell or tissue type. Real-time PCR is often marketed as RT-PCR, it should not be confused with reverse transcription polymerase chain reaction also known as RT-PCR.
The procedure follows general principle of PCR Two common methods of quantification Fluorescent dyes that intercalate with double-stranded DNA (SYBR Green) modified DNA oligonucleotide probes that fluoresce when hybridized with a complementary DNA (TaqMan probe)
SYBR Green dye A DNA-binding dye binds to all dsdna in a PCR reaction causing fluorescence of the dye. Increase in DNA product during PCR, leads to an increase in fluorescence intensity and is measured at each cycle, thus allowing DNA concentrations to be quantified. bind to all dsdna PCR products, including nonspecific PCR products (such as "primer dimers"). This can potentially interfere with or prevent accurate quantification of the intended target sequence.
SYBR Green dye
TaqMan probe Two types of fluorophores Q reduces the fluorescence of R whenever the probe attached or unattached to DNA template, before polymerase acts. Does this by FRET (Fluorescence Resonance Energy Transfer)
TaqMan probe TaqMan probe binds to specific DNA template after denaturation. Reaction cool and primer anneals to the DNA. Polymerase collides with TaqMan probe
TaqMan probe Taq polymerase adds nucleotides and removes TaqMan probe from DNA template So separates R from Q and allow R to emit its energy This emission signal then quantified with computer.
Quantitation
Increase in Reporter signal The increase in reporter signal is captured by the Sequence Detection instrument and displayed by the software. Figure shows an increase in the reporter signal over time. The amount of reporter signal increase is proportional to the amount of product being produced for a given sample.
Amplification curve A threshold for detection of fluorescence above background is determined. The cycle at which the fluorescence from a sample crosses the threshold is called the cycle threshold, Ct. Quantity of DNA doubles every cycle during the exponential phase, relative amounts of DNA can be calculated, e.g. a sample whose Ct is 3 cycles earlier than another's has 23 = 8 times more template.
PCR phases Three replicates of sample with same starting material. Amplification occurs exponentially because all reagents are fresh and available. Reactions start to slow down in Linear phase. Reactions stop at end-point (plateau)
PCR phases Linear view
PCR phases Log view
PCR phases Linear view in 96 replicates The reactions show a clear separation at the plateau phase Therefore, if the Therefore, if the measurements were taken in the plateau phase, quantitation would be affected.
PCR phases Log view in 96 replicates The plateau of each reaction seems to occur in the same place. However, this is solely due to the log scaling of the plot.
PCR phases Log view 5-fold dilution series The 5-fold dilution series, seems to plateau at the same place even though the exponential phase clearly shows a difference between the points along the dilution series. This reinforces the fact that if measurements were taken at the plateau phase, the data would not truly represent the initial amounts of starting target material.
Normal PCR Difficult to differentiate 5-fold change in agarose gel.
Amounts of RNA or DNA are then determined by comparing the results to a standard curve produced by RT-PCR of serial dilutions (e.g. undiluted, 1:4, 1:16, 1:64) of a known amount of RNA or DNA. To accurately quantify gene expression, the measured amount of RNA from the gene of interest is divided by the amount of RNA from a housekeeping gene measured in the same sample to normalize for possible variation in the amount and quality of RNA between different samples. Normalization permits accurate comparison of expression of the gene of interest between different samples, provided that the expression of the reference (housekeeping) gene used in the normalization is very similar across all the samples.
Issues addressed. Optimized the reagents and conditions used in the amplification reaction. Carry out calibration experiments in which progressively smaller amounts of standard templates are amplified. Establish the reproducibility of the methods by performing a statistical significant number of multiple assays on the same sets of templates and, finally, on independently processed samples. Perform control experiments showing that the efficiency of amplification of target and reference molecules are equal during the course of the PCR.
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