real time manudagur(2)

7/31/2019 Real Time Manudagur(2)

1/31

Real-Time PCRReal-Time PCRmRNA quantification


2/31

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)


3/31

quantitative mRNA/DNA analysis

Direct

-Northern blotting

-In situ hybridization

PCR amplification

-Regular RT-PCR-Real time PCR

(Microarrays)


4/31

Nomenclature

RT-PCR = Reverse Transcriptase PCR

qReal time PCR = quantitative Real-Time PCR


5/31

RT-PCR

Isolate RNA

cDNA synthesis

PCR reaction


6/31

Why isnt this good enough?


7/31

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)
http://www.appliedbiosystems.com/support/tutorials/pdf/rtpcr_vs_tradpcr.pdfhttp://www.appliedbiosystems.com/support/tutorials/pdf/rtpcr_vs_tradpcr.pdf


8/31

Different concentrations give similar

endpoint results!

Endpoint analysis


9/31

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


10/31

Polymerization

3QR ProbeForward

Primer

3 5

5 3

Reverse

Primer

5

5

R = Reporter

Q = Quencher


11/31

For Real Time PCR we need a

a specific probe with afluorescent reporter.

R QProbe


12/31

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


13/31

StrandDisplacement

3Q3 5

5 3

5

5

R


14/31

Cleavage

3Q

R

3 5

5 3

5

5


15/31

Polymerization Completed

3QR

3 5

5 3

5


16/31

Different concentrations give similar

endpoint results!

Endpoint analysis


17/31

Van der Velden. Leukemia 2003 (www)
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=12764363http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=12764363


18/31

SYBR Green(double-stranded DNA binding dye)

* emits a strong fluorescent signal upon binding todouble-stranded DNA

* nonspecific binding is a disadvantage

* requires extensive optimisation

longer amplicons create a stronger signal

Its cheap


19/31

Forward

Primer

3' 5'

5' 3'

Reverse

Primer

5'

5'

Polymerization

3' 5'

5' 3'

5'

5'

Polymerization completed

SYBR Green I Chemistry


20/31

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


21/31


22/31

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)


23/31

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


24/31

Van der Velden. Leukemia 2003 (www)
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=12764363http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=12764363


25/31

(www)
http://www.ambion.com/http://www.ambion.com/


26/31

(www)
http://www.ambion.com/http://www.ambion.com/


27/31

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 haschanged 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 anothergene which expression is normally constant, a housekeepinggene


28/31

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


29/31

Pure Dyes

Wavelength (nm)500nm 660nm


30/31

What is Multiplexing?


31/31

Real-Time PCR Applications

* quantitation of gene expression* drug therapy efficacy / drug monitoring

* viral quantitation

* pathogen detection

real time manudagur(2)

Documents