real time rt-pcr quantitating gene expression
TRANSCRIPT
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Real time RT-PCR
Quantitating Gene Expression
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Real-time PCR monitors the fluorescence emitted duringthe reaction as an indicator of amplicon production ateach PCR cycle (in real time) as opposed to the endpointdetection
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•Based on the detection and quantitation of a fluorescent reporter
* The first significant increase in the amount of PCR product (CT - threshold cycle) correlates to the initial amount of target template
Real-time Principles
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Amplification Plots
Gestl 082709b.mxp
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Amplification Plots
Gestl 082709b.mxp
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Real-Time Principles
Three general methods for the quantitative assays:
1. Hydrolysis probes(TaqMan, Beacons, Scorpions)
2. Hybridization probes(Light Cycler)
3. DNA-binding agents(SYBR Green)
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Multiplexing: TaqMan: Yes, different dyes for each target (FAM, TET, VIC and JOE)SYBR Green: No
Cost: TaqMan: More expensive when doing multiple genesSYBR green: Less expensive
Specificity: TaqMan: More specificSYBR green: Less specific
Advantages/Disadvantages
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Threshold Cycle
• Threshold cycle or the CT value is the cycle at which a significant increase in ∆Rn is first detected
• Parameter used for quantitation
• CT value of 40 or more means no amplification and cannot be included in the calculations
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Amplification Plots
Gestl 082709b.mxp
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Amplification Plots
Gestl 082709b.mxp
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Standard Curve
Gestl 082709b.mxp
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* Rn+ is the Rn value of a reaction containing allcomponents (the sample of interest); Rn- is the Rnvalue detected in NTC (baseline value)* ∆Rn is the difference between Rn+ and Rn-. It is anindicator of the magnitude of the signal generated bythe PCR* ∆Rn is plotted against cycle numbers to produce theamplification curves and gives the CT value
What is ∆Rn?
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What is ∆Rn?
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Control Amplification Plots
Gestl 061009.mxpPol delta 2
No RT
No TC
No PC
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Amplification Plots of Pol beta
No RT Controls
Gestl 061009.mxp
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One-Step vs. Two Step Reactions
•One-step real-time RT-PCR performs reverse transcription and PCR in a single buffer system and in one tube
• Two-step RT-PCR, performs the reactions separately in different tubes
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Endogenous/Internal Control(Normalization)
•Usually an abundantly and constantly expressed housekeeping gene
•Most commonly used ones are the least reliable ones
•Best to run a validity test for the selected endogenous control
* Combination may/should be used
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Absolute Quantification
-Determine the actual number of molecules in the reaction
-Comparison to a Standard
-Each Standard is unique
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Dissociation Curve of Pol delta 2
Gestl 061009.mxp
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Primer Concentration of 6-day Samples
Gestl 061009.mxpPol delta 2
300 – 300 nM
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Problems & SolutionsProblems:
-Multiple peaks in Dissociation Curves-Product in No Template control-Product in No RT Control
Solution:-Design new primers
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Standard Curve
Gestl 082709b.mxp
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Amplification Plots
Gestl 082709b.mxp