Real-Time PCR mRNA quantification

What do mRNA levels tell us? DNA  mRNA  protein 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) (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 3 Q R Probe Forward Primer Reverse Primer 5 5 R = Reporter Q = Quencher

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

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

Strand Displacement 3 Q R

Cleavage 3 Q R

Polymerization Completed 3 Q R

Different concentrations give similar endpoint results! Endpoint analysis

Van der Velden. Leukemia 2003 (www) (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' 3' Reverse Primer 5' Polymerization 3'5' 3' 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 C T value is the cycle at which a significant increase in Rn is first detected * it is the parameter used for quantitation * C T value of 40 or more means no amplification and cannot be included in the calculations Data analysis

Van der Velden. Leukemia 2003 (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 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 Wavelength (nm) 500nm660nm

What is Multiplexing?

Real-Time PCR Applications * quantitation of gene expression * drug therapy efficacy / drug monitoring * viral quantitation * pathogen detection