Real time RT-PCR

Quantitating Gene Expression

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

•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

Amplification Plots

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Amplification Plots

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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)

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

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

Amplification Plots

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Amplification Plots

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Standard Curve

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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?

What is ∆Rn?

Control Amplification Plots

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No RT

No TC

No PC

Amplification Plots of Pol beta

No RT Controls

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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

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

Absolute Quantification

-Determine the actual number of molecules in the reaction

-Comparison to a Standard

-Each Standard is unique

Dissociation Curve of Pol delta 2

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Primer Concentration of 6-day Samples

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300 – 300 nM

Problems & SolutionsProblems:

-Multiple peaks in Dissociation Curves-Product in No Template control-Product in No RT Control

Solution:-Design new primers

Standard Curve

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Amplification Plots

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