Real-Time PCR The Polymerase Chain Reaction (PCR) is a process for the amplification of specific fragments of DNA. Real-Time PCR a specialized technique that allows a PCR reaction to be visualized “in real time” as the reaction progresses. Real-Time PCR allows us to measure minute amounts of DNA sequences in a sample. Uses of Real-Time PCR Real-Time PCR has become a cornerstone of molecular biology: Gene expression analysis Cancer research Drug research Disease diagnosis and management Viral quantification Food testing Testing of GMO food Animal and plant breeding Gene copy number

1. Real Time PCR
Submitted by: Darakhshan Fatima
1

2. Real-Time PCR
The Polymerase Chain Reaction (PCR) is a process for the
amplification of specific fragments of DNA.
Real-Time PCR a specialized technique that allows a PCR reaction
to be visualized “in real time” as the reaction progresses.
Real-Time PCR allows us to measure minute amounts of DNA
sequences in a sample.
2

3. Uses of Real-Time PCR
Real-Time PCR has become a cornerstone of molecular biology:
• Gene expression analysis
Cancer research
Drug research
• Disease diagnosis and management
Viral quantification
• Food testing
Percent GMO food
• Animal and plant breeding
Gene copy number
3

4. Instrumentation of Real-Time
PCR
Real-time PCR instruments consist of THREE main components:
1. Thermal Cycler (PCR machine)
2. Optical Module (to detect fluorescence in the tubes during the run)
3. Computer (to translate the fluorescence data into meaningful
results)
4

5. 2a. excitation
filters
2b. emission
filters
1. halogen
tungsten lamp
4. sample plate
3.
intensifier
5.detec
tor
350,000
pixels
Fig 1: Instrumentation of qPCR
Source: http://www.bio-rad.com/en-uk/applications-technologies/introduction-qpcr-instrumentation?ID=LUSO5YMNI
5

6. Fig 2: Bio-rad iQ5 Real Time PCR
Source:http://scientificentre.com/products.php?product=Bio%252dRad-iCycler-Thermal-Cycler-iQ5-
Multicolor-Real%252dTime-PCR-Detection-System

7. Steps in Real-time PCR
There are three major steps that make up each cycle in a real-time
PCR reaction. Reactions are generally run for 40 cycles.
1. Denaturation
• High temperature incubation is used to “melt” double-stranded
DNA into single strands and loosen secondary structure in
single stranded DNA.
• The highest temperature that the DNA polymerase can
withstand is typically used (usually 95°C).
• The denaturation time can be increased if template GC
content is high.
7

8. 2. Annealing
• During annealing, complementary sequences have an
opportunity to hybridize, so an appropriate temperature is
used that is based on the calculated melting temperature (Tm)
of the primers (5°C below the Tm of the primer).
3. Extension
• At 70-72°C, the activity of the DNA polymerase is optimal,
and primer extension occurs at rates of up to 100 bases per
second.
• When an amplicon in real-time PCR is small, this step is often
combined with the annealing step using 60°C as the
temperature.
8

9. cDNA
Syber
green
Master
Mix
Forward
and
reverse
primers
Nuclease
free
water
House
keeping
gene
Major Components and
Parameters of Real-time PCR
9

10. Primer design considerations
The following recommendations are offered for designing
primers for real-time PCR:
• In general, design primers that are 18–28 nucleotides in length
• Avoid stretches of repeated nucleotides
• Aim for 50% GC content, which helps to prevent mismatch
stabilization
10

11. Real-time PCR fluorescence
detection systems
SYBR Green dye
• SYBR Green I dye is a fluorescent DNA binding dye, binding
to the minor groove of any double-stranded DNA.
• Excitation of DNA-bound SYBR Green dye produces a much
stronger fluorescent signal compared to unbound dye.
11

12. Fig 2: Steps of Real time PCR
Source: https://www.researchgate.net/figure/Outline-of-different-Steps-of-a-Quantitative-Real-
12

13. PCR Plates
Fig 2: PCR plate
Source: https://www.camlab.co.uk/brand-pcr-plates-p13953.aspx
13

14. • 48-well plates, 96-well plates, 384-well plates,
3072-through-hole plates, etc are available.
• It includes following four steps:
Plate
Preparation
Plate
Loading
Plate
Insertion
Plate
Sealing
14

15. Plate Preparation
• Mix all PCR components before starting reaction
• Gently swirl enzyme-containing master mixes
• Briefly (1–2 seconds) vortex other components such as PCR
primer.
• Thaw nucleic acid samples.
• Gently inverting tubes a few times or lightly vortexing for 1-2
seconds are highly efficient mixing methods.
• Centrifuge briefly to collect the contents at the bottom of the
container and eliminate any air bubbles from the solutions
15

16. Plate loading
• Plan your pipetting to avoid cross contamination of samples
and assays.
• Make a master mix.
• Mixing fully assembled reactions is not necessary, because
nucleic acids are hydrophilic and will quickly mix with the
assay master mix.
16

17. Plate sealing
• Plastic PCR plates may be sealed with optical caps or optical
covers, which are thin sheets of plastic with adhesive on one
side.
• Once the plate has been sealed, hold it up and inspect the
bottom, looking for any anomalies, such as unintended empty
wells, drops of liquid adhering to the walls of the well, and air
bubbles at the bottom of the well.
• Centrifuge the plate briefly to correct any adherent drop and
bottom bubble problems.
• Plates may be safely labeled along the skirt.
• Do not write on the surface of the plate over well positions,
as this will interfere with fluorescence excitation and reading.
• Significant block contamination by colored or fluorescent
substances can adversely affect real-time PCR data. 17

18. Plate insertion
• Once the plate is ready, it may be loaded into the real-time
PCR instrument, following the manufacturer’s instructions.
• Plates containing DNA or cDNA template and reagents are
very stable at ambient temperature.
• They can be routinely stored at room temperature under
normal laboratory lighting for days without ill effect.
• Avoid direct exposure of loaded plates to sunlight; this can
compromise the fluorescent dyes in the mixture.
• If you need to transport a plate outside, wrap the plate in
aluminum foil to protect it from sunlight.
18

19. Contamination
Some of the possible sources of contamination are:
• Cross-contamination between samples
• Contamination from laboratory equipment
• Carryover contamination of amplification products and
primers from previous PCRs.
• This is considered to be the major source of false positive PCR
results
19

20. Prevention
• Uracil DNA glycosylase (UDG) is used to reduce or prevent
DNA carryover contamination between PCR reactions by
preventing the amplification of DNA from previous reactions.
20

21. Internal controls and reference
genes
• It is critical to use reliable internal control gene products for
the normalization of expression levels between experiments.
• Relative gene expression comparisons work best when the
expression level of the chosen housekeeping gene remains
constant.
21

22. Endogenous Controls
• β-actin (BACT): cytoskeletal gene
• 18S Ribosomal RNA (rRNA): ribosomal subunit
• Cyclophilin A (CYC): serine-threonine phosphatase inhibitor
• Glyceraldehyde phosphate dehydrogenase (GAPDH):
glycolysis pathway
• β-2-microglobulin (B2M): major histocompatibility complex
• TATA-Box binding protein (TBP): RNA transcription
22

23. Data Analysis in Real-time PCR
• It is achieved by the following methods:
Absolute Quantification
• This is achieved by comparing the CT values of the test
samples to a standard curve.
• The result of the analysis is quantity of nucleic acid (copy
number, µg) per given amount of sample (per cell, per µg of
total RNA).
Relative Quantification
• In relative quantification, the analysis result is a ratio: the
relative amount (fold difference) of a target nucleic acid for
equivalent amounts of test and control sample A vs. B.
• Both cases need to address the question of what the “amount
of sample” is, and in relative quantification, to ensure that
equivalent amounts of samples are compared.
23

24. Disadvantages
of qPCR
Costly
Equipment
High cost of
chemicals
Complicated
Data Analysis
and
Interpretation
Highly
sensitive
Reaction
Disadvantages of Real time PCR
24

25. Advantages of qPCR
Speed
Sensitive
reaction
Highly
reproducible 25

26. References
• Chen, C., Ridzon, D. A., Broomer, A. J., Zhou, Z., Lee, D. H., Nguyen, J.
T., & Lao, K. Q. 2005. Real-time quantification of microRNAs by stem–
loop RT–PCR. Nucleic acids research,33(20): e179-e179.
• Klein, D.2002. Quantification using real-time PCR technology:
applications and limitations. Trends in molecular medicine, 8(6): 257-260.
• Schmittgen, T. D., & Livak, K. J. 2008. Analyzing real-time PCR data by
the comparative C T method. Nature protocols,3(6): 1101.
• Vandesompele, J., De Preter, K., Pattyn, F., Poppe, B., Van Roy, N., De
Paepe, A., & Speleman, F. 2002. Accurate normalization of real-time
quantitative RT-PCR data by geometric averaging of multiple internal
control genes.Genome biology, 3(7): research0034-1.
• Valasek, M. A., & Repa, J. J. 2005. The power of real-time PCR. Advances
in physiology education, 29(3): 151-159.
• Yuan, J. S., Reed, A., Chen, F., & Stewart, C. N. 2006. Statistical analysis
of real-time PCR data. BMC bioinformatics,7(1): 85.
• Wong, M. L., & Medrano, J. F.2005. Real-time PCR for mRNA
quantitation. Biotechniques, 39(1): 75-85.
26