Real-Time PCR

mRNA quantification

What do mRNA levels tell us?

DNAmRNAprotein Reflect level of gene expression Information about cell response Protein production (not always)

quantitative mRNA/DNA analysis

Direct -Northern blotting -In situ hybridization PCR amplification -Regular RT-PCR -Real time PCR (Microarrays)

Nomenclature

RT-PCR = Reverse Transcriptase PCR qReal time PCR = quantitative Real-Time PCR

RT-PCR

Isolate RNA cDNA synthesis PCR reaction

Why isnt this good enough?

Whats Wrong With Agarose Gels?

* * * * *

Low sensitivity Low resolution Non-automated Size-based discrimination only Results are not expressed as numbers based on personal evaluation Ethidium bromide staining is not very quantitative End point analysis
ABI: Real-Time PCR vs Traditional PCR (www)

Endpoint analysis

Different concentrations give similar endpoint results!

Real-time Principles
based on the detection and quantitation of a fluorescent reporter In stead of measuring the endpoint we focus on the first significant increase in the amount of PCR product. The time of the increase correlates inversely to the initial amount of DNA template

Polymerization
Forward Primer

5 3 5

Probe

R = Reporter Q Q = Quencher
3 5 3 5

Reverse Primer

For Real Time PCR we need a a specific probe with a fluorescent reporter.
R

Probe

When in close contact with the reporter, the quencer absobes its emission.

Strand Displacement
R
5 3 5

3 5 3 5

Cleavage
R
5 3 5

3 5 3 5

Polymerization Completed
R
5 3 5

5 3

Endpoint analysis

Different concentrations give similar endpoint results!

Van der Velden. Leukemia 2003 (www)

(double-stranded DNA binding dye) * emits a strong fluorescent signal upon binding to double-stranded DNA * nonspecific binding is a disadvantage * requires extensive optimisation longer amplicons create a stronger signal Its cheap

SYBR Green

SYBR Green I Chemistry

Polymerization
5' 3' 5' Reverse Primer Forward Primer 5' 3' 5'

Polymerization completed
5' 3' 5' 5' 3' 5'

Real-time PCR advantages

* not influenced by non-specific amplification * amplification can be monitored real-time * no post-PCR processing of products
(high throughput, low contamination risk) (3 picogram = one genome equivalent)

* requirement of 1000-fold less RNA than conventional assays * most specific, sensitive and reproducible

Real-time PCR disadvantages

* setting up requires high technical skill and support * high equipment cost * Runs are more expensive than conventional PCR * DNA contamination (in mRNA analysis)

Data analysis
Cycle Threshold
* cycle threshold or the CT value is the cycle at which a significant increase in Rn is first detected * it is the parameter used for quantitation * CT value of 40 or more means no amplification and cannot be included in the calculations

Van der Velden. Leukemia 2003 (www)

(www)

(www)

Housekeeping gene
Knowing the amount of mRNA in one sample from one specific gene does not tell us alot You dont know the total amount of mRNA in your sample You also dont know how much the mRNA level has changed compared to other mRNA levels Example: mRNA levels increase 2x after induction It is possable that all genexpression in the cell has increased We have to compare the expression of our gene to another gene which expression is normally constant, a housekeeping gene

Multiplexing
* TaqMan: different dyes for each target (FAM, TET, VIC and JOE) * SYBR green: different melting points for each target * extensive optimisation is required * one-step PCR cannot be used

Pure Dyes

500nm

Wavelength (nm)

660nm

What is Multiplexing?

Real-Time PCR Applications

* quantitation of gene expression * drug therapy efficacy / drug monitoring * viral quantitation * pathogen detection