Real-Time PCR DNA quantification Prepared: Alibekova A. Group: BT 16 -02
What do m. RNA levels tell us? DNA m. RNA protein • Reflect level of gene expression • Information about cell response • Protein production (not always)
quantitative m. RNA/DNA analysis Direct -Northern blotting -In situ hybridization PCR amplification -Regular RT-PCR -Real time PCR (Microarrays)
Nomenclature RT-PCR = Reverse Transcriptase PCR q. Real time PCR = quantitative Real-Time PCR
RT-PCR • Isolate RNA • c. DNA 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)
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 5 3 Forward Primer R Probe R = Reporter Q Q = Quencher 3 5 5 Reverse Primer 3 5
For Real Time PCR we need a a specific probe with a fluorescent reporter. R Probe Q
When in close contact with the reporter, the quencer absobes its emission.
Strand Displacement R 5 3 5 Q 3 5
Cleavage R 5 Q 3 3 5 5 3 5
Polymerization Completed R Q 3 5 5 3
Endpoint analysis Different concentrations give similar endpoint results!
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
SYBR® Green I Chemistry Polymerization 5' Forward Primer 3' 5' 5' 3' Reverse Primer 5' Polymerization completed 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) * 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 m. RNA analysis)
Data analysis Cycle Threshold * cycle threshold or the CT value is the cycle at which a significant increase in DRn 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)
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Housekeeping gene • Knowing the amount of m. RNA in one sample from one specific gene does not tell us alot • You don´t know the total amount of m. RNA in your sample • You also dont know how much the m. RNA level has changed compared to other m. RNA levels Example: m. RNA levels increase 2 x 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 * Taq. Man: 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 500 nm Wavelength (nm) 660 nm
What is Multiplexing?
Real-Time PCR Applications * quantitation of gene expression * drug therapy efficacy / drug monitoring * viral quantitation * pathogen detection
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