Real-time PCR/quantitative PCR

HUIMM906 – Spring 2017

 What is qPCR?

• Lab technique: molecular biology

• Based on PCR:
– Conventional – end point detection

• Real time recording of DNA amplification

– Fluorescence intensity

• Determine the starting concentration of nucleic acid

 Traditional PCR…

https://www.thermofisher.com/no/en/home/life-science/pcr/real-time-pcr.html
https://bioprep.community.uaf.edu/files/2013/07/gel1.jpg
 …vs qPCR

• Exponential phase

• Threshold
(fluorescent
intensity above
background)

• Threshold cycle
(CT): the PCR cycle
at which the sample
reaches the
threshold

https://www.thermofisher.com/no/en/home/life-science/pcr/real-time-pcr.html
Visualization methods
 TaqMan and SYBR Green comparison

TaqMan SYBR Green

fluorogenic probe: detects a specific PCR double stranded DNA binding dye:
product detects all double stranded DNA (specific
and nonspecific)

(+) specific hybridization between probe (+) no probes required (running costs
and target (reduced background and false reduced)
positives)

(+) multiplex PCR (-) false positives

(-) different probes synthesized for each (-) primer optimization needed
unique target sequence
 Experimental design
• Identify target of interest
– One (singleplex - SYBR Green)…
– … or more (multiplex - TaqMan)
• Probes: different fluorescent dyes

• Endogenous controls
– Differences in sampling
– Avoid misinterpretation of results
– Normalization: housekeeping genes (GAPDH, actin etc)
 Detection methods
• Thermal cycler with fluorescent detection and
specialized software

https://shop.roche.com/wcsstore/RASCatalogAssetStore/Products/3.8.1.4.1.3/Images/3.8.1.4.1.3-large.jpg
http://www.sabiosciences.com/pathwaymagazine/img/pathways7/page13a.jpg
Quantification methods

Relative
• Differences in expression level of target gene between different samples
• treated vs untreated
• brain vs liver
• drug treatment after 2-6-12 hr

Absolute
• Standard curve method: dilution series of known copy numbers
 An easy example…
Stage Sample 1 Sample 2
To begin with: 2 copies of gene of interest 6 copies of gene of interest
(maybe treated)
1st cycle 4 12
2nd cycle 8 24
3rd cycle 16 48
We have awesome detector! (>45) CT=3
4th cycle 32 96
5th cycle 64 192
CT=4.6 We have awesome detector! (>45) mmmmmm

CT (S1)=4.6 CT of S2 precedes that of S1 by 1.6 cycles

CT (S2)=3 So, S2 contained 21.6≈3 more template than S1
 Another easy example…
6
Stage Std1 Std2 Std3 Std4 Std5 Sample
5 begin 4 8 16 32 64 x
1st cycle 8 16 32 64 128 2x
4
Detector >100 CT=1

3
2nd cycle 16 32 64 128 256 4x
Detector >100 CT=2
2 3rd cycle 32 64 128 256 512 8x
CT=3
1
4th cycle 64 128 256 512 1024 16x

0 CT=4 CT=4
0 1 2 3 4 5

The efficiency of amplification is variable

 Our (fake) results
Sample Initial copy number CT
Std1 100 30.79
Std2 1.000 26.68
Std3 10.000 -
Std4 100.000 18.68
Std5 1.000.000 15.43
sample unknown 21.92

Crossing point = -3.794 x log concentration + 37.97

Unknown sample: crossing point = 21.92

Log concentration = 4.23
Unknown sample concentration = 104.23 = 16.982,4 = 1.7x104
 What can you do with qPCR?
• Gene expression analysis: mRNA

https://www.saylor.org/content/bio403/assessments/RT.gif
 What can you do with qPCR?
• diagnostics:
– infectious diseases (nucleic acids of pathogens: HepB)
– cancer/genetic abnormalities (known transcripts, unique
splice variant, homologs and orthologs)
• microbiology: food safety, food spoilage,
fermentation, microbial risk assessment of water
quality
• research: gene transcription over time (cell
differentiation), in different tissues, after drug
administration
• GMO (more than 1 copy of the transgene)
Good luck with your experiments!