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mRNA quantification
Nomenclature
RT-PCR = Reverse Transcriptase PCR qReal time PCR = quantitative Real-Time PCR
RT-PCR
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
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
(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
Polymerization completed
5' 3' 5' 5' 3' 5'
* requirement of 1000-fold less RNA than conventional assays * most specific, sensitive and reproducible
* 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
(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?