Real-time PCR

April 10, 2007 FW5080

Gene Expression Analysis

Conventional Methods
Northern RT-PCR Semi-quantitative RT-PCR (sub-sampling at various cycles)

Problems:
Labor intensive Hazardous materials (e.g., 32P) Low resolution & sensitivity Low dynamic range Poor discrimination among homologous genes or transcript sizes Results not expressed as numbers Not very quantitative

Conventional RT-PCR vs. Real-time RT-PCR

Conventional RNA extraction Real-time RNA extraction DNase treatment
(random vs. oligo dT primers)

Reverse transcription

(random vs. oligo dT primers)

Different chemistries: PCR DNA binding dyes (40 cycles, with 2.5-10 ng cDNA) Hybridization probes Hydrolysis probes Hairpin probes

Reverse transcription

(25-35 cycles, with 25-100 ng cDNA)

PCR

Gel electrophoresis
Southern hybridization

Data analysis & quantification

Image analysis & quantification

PCR Amplification Curve

Plateau phase

Log-linear phase

Linear ground phase Early exponential phase

PCR Amplification Curve

Conventional RT-PCR & gel electrophoresis

CT = the PCR cycle in which the fluorescence intensity generated by the accumulation of specific products is significantly higher (10X SDs of the baseline mean) than the background fluorescence

Semi-quantitative RT-PCR with multiple sampling & gel electrophoresis

10 5 2.5 1.25 0.625 ng

Threshold fluorescence

Early exponential phase

Cycle of threshold (Ct) 24.8

26.8 29.2 25.8 28

Real-time PCR Detection Chemistries

1. DNA-binding dyes (e.g., SYBR Green)
Emit fluorescence when bound to dsDNA Fluorescence intensity ~ dsDNA concentration

Advantages
Flexible, compatible with existing RT-PCR primers Relatively low costs

Disadvantages
Dye binding not sequence-specific (false positives)
Dissociation curve analysis necessary

Multiplexing not possible A standard curve is required for absolute quantification, otherwise measurement is relative only.

Wong & Medrano (2005) BioTechniques 39: 75-85. van der Velden et al. (2003) Leukemia 17:1013-34.

Dissociation curve or melting curve analysis to distinguish specific vs. non-specific products

Real-time PCR Detection Chemistries

2. Hybridization probes
2 PCR primers, and 2 gene-specific probes that bind adjacent to each other the upstream probe is labeled with an acceptor dye on the 3-end, and the downstream one with a donor dye at the 5-end (bkgd emission). When bound, FRET (fluorescence resonance energy transfer) increased fluorescence Fluorescence intensity correlates inversely with PCR product accumulation, quantification by melting curve analysis

Pros and Cons

Allele-specific discrimination Multiplex, though possible, is challenging
Wong & Medrano (2005) BioTechniques 39: 75-85. Bustin (2000) J Molecular Endocrinology 25: 169193.

Real-time PCR Detection Chemistries

3. Hydrolysis probes (e.g., TaqMan)
Based on Taq 5-exonuclease activity (5 nuclease assay) A sequence-specific probe is labeled with a reporter dye on the 5 end and a quencher dye on the 3 end. When the probe is intact, the quencher reduces the reporter fluorescence intensity by FRET (bkgd emission). When annealed to the template, the bound and quenched probe will be degraded by Taq 5-exonuclease activity during PCR extension Hydrolysis leads to separation of reporter & quencher increased reporter fluorescence emission
Annealing phase

Extension phase (I)

Extension phase (II)

Pros and Cons

Sequence-specific detection, multiplexing Probe design may be challenging Probes are expensive Extensive optimization may be required
End of PCR cycle

Wong & Medrano (2005) BioTechniques 39: 75-85. van der Velden et al. (2003) Leukemia 17:1013-34.

Real-time PCR Detection Chemistries

4. Hairpin probes (e.g., Molecular beacons)
The probe consists of a sequence-specific region (loop) flanked by 2 inverted repeats, labeled with reporter and quencher dyes. In its hairpin configuration, FRET reduces the reporter fluorescence When annealed to the target, the quencher and reporter are separated increased reporter fluorescence emission

Pros and Cons

Sequence-specific detection, multiplexing Greater specificity than linear probes Probe design may be challenging Probes are expensive Extensive optimization may be required
Wong & Medrano (2005) BioTechniques 39: 75-85. http://www.biolegio.com/realtimepcr/molecularbeacon/

SYBR Green Real-time PCR Considerations

DNase treatment recommended (but not the entire RNA stock) RT: oligo dT or random-priming (see previous notes) Primer design: targeting 3-UTR, 150-300 bp amplicons
Intron-flanking primers ( genomic DNA contamination)
RTPrimerDB (http://medgen.ugent.be/rtprimerdb/) Primer Bank (http://pga.mgh.harvard.edu/primerbank/index.html)

Housekeeping genes:
Require empirical determination Recommend >3 if comparing across multiple tissue types (Vandesompele et al., Genome Biology, 2002); 1 maybe ok for single tissue comparison Use geometric mean as normalization basis

Absolute vs. relative quantification, what do you need?

Amplification efficiency Standard curve

Relative Quantification Methods

Determines changes in steady-state mRNA levels of the gene of interest (GOI) across multiple samples, and expresses it relative to the levels of HKG(s). Does not require a standard curve, adequate for most purposes.

1. CT method
CT inversely correlates with starting transcript copies Amplicons double after each cycle (assuming 100% efficiency) 1 cycle difference = 2-fold difference in transcript abundance Relative fold change = 2CT (CT= 4.2, relative difference= 18.4X) CT = [CT of GOI] [geomean of CT of HKGs] Amplification efficiency may vary between genes and samples!

Tevfik DORAK website, http://www.gene-quantification.de

Effects of PCR Amplification Efficiency

Exponential amplification = 10(-1/slope) PCR Efficiency = 10(-1/slope) 1
Pfaffl (2001) Nucleic Acids Research 29:e45

PCR amplification = (1+E)n, n=cycle Starting w/ 1 molecule, after n=30: (1) E=0.9 1x(1+0.9)30= 2.3x108 (2) E=0.8 1x(1+0.8)30= 4.6x107

Should cover experimental range Needed for every GOI and HKG

Stratagene: dilution series (1:5), X 6 points (n=3) Acceptable efficiency = 10010%

Tevfik DORAK website, http://www.gene-quantification.de

Relative Quantification Methods

Determines changes in steady-state mRNA levels of the gene of interest (GOI) across multiple samples, and expresses it relative to the levels of HKG(s). Does not require a standard curve, adequate for most purposes.

2. CT method
First normalize the GOI expression level to HGK(s) CT Then compare it to a control sample (calibrator) CT Relative expression = 2-CT Applicable for treated vs. non-treated comparisons; not applicable for gene expression analysis across different tissues Several software packages available (geNom, qBASE, REST, etc)

Emerging trend: computing efficiency from individual PCR rx. Efficiency may be affected by:
- sample degradation, amplicon size, PCR condition, human errors,
Tevfik DORAK website, http://www.gene-quantification.de