Real-Time PCRReal-Time PCRmRNA quantification

What do mRNA levels tell us?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 isn´t this good enough?

What’s 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)

Different concentrations give similar endpoint results!

Endpoint analysis

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

3QR Probe

Forward Primer

3 5

5 3

Reverse Primer

5

5

R = ReporterQ = Quencher

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

R QProbe

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

Strand Displacement

3Q

3 5

5 3

5

5

R

Cleavage

3Q

R

3 5

5 3

5

5

Polymerization Completed

3QR

3 5

5 3

5

Different concentrations give similar endpoint results!

Endpoint analysis

Van der Velden. Leukemia 2003 (www)

SYBR Green (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

• It´s cheap

Forward Primer

3' 5'

5' 3'

Reverse Primer

5'

5'

Polymerization

3' 5'

5' 3'

5'

5'

Polymerization completed

SYBR® Green I Chemistry

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)

* requirement of 1000-fold less RNA than conventional assays(3 picogram = one genome equivalent)

* 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)

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

Data analysis

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 don´t know the total amount of mRNA in your sample• You also dont know how much the mRNA level has

changed compared to other mRNA levelsExample:

mRNA levels increase 2x after inductionIt is possable that all genexpression in the cell has increasedWe 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

Wavelength (nm)500nm 660nm

What is Multiplexing?

Real-Time PCR Applications

* quantitation of gene expression

* drug therapy efficacy / drug monitoring

* viral quantitation

* pathogen detection