This document provides an overview of real-time PCR. It discusses the basics of real-time PCR including how it works, terminology, and uses. It also covers experimental design considerations like singleplex vs multiplex assays and fluorescent chemistry selection. Practical aspects are reviewed like plate loading, standard curves, and melting curves. Data analysis techniques like absolute and relative quantification and gene expression analysis are also summarized.

1. REAL TIME PCR
T R A I N I N G C O U R S E BY
D R . M U S L I M D. M U S A

2. IN THIS TRAINING
1- basics of real time PCR
• Definition
• Real-Time PCR vs Conventional PCR
• How real time PCR works
• Real-Time PCR terminology
• Uses of Real-Time PCR
2-Experimental design
• Singleplex and multiplex
• Selection of Real-Time chemistry
3- Practical part
• Plate loading
• Standard curve preparation
• Melting curve
4-Data analysis
• Absolute quantification
• Relative quantification
• Gene expression analysis
• Allelic discrimination

3. EVERY POLYMERASE CHAIN REACTION MUST HAS
The polymerase chain reaction (PCR) is one of the most powerful
technologies in molecular biology.
Using PCR, specific sequences within a DNA or cDNA template can be
copied, or “amplified”, many thousand- to a million-fold
By using:
Sequence specific oligonucleotides (primers /probe)
Heat stable DNA polymerase.
Thermal cycling.

5. BASIC IDEA OF REAL TIME PCRGenerally polymerase chain reaction (PCR) is a mean for detection and amplification of
nucleic acid (DNA).
In conventional PCR, only tell you there is nucleic acid of interest or not but it can not tell
you how much DNA present(copy number) in this sample. ( how much starting material
????)
To known the concentration of starting materials, scientists reasoned that the number of
cycles and the amount of PCR end-product could be used to calculate the initial quantity
of genetic material by comparison with a known standard.
Real-time PCR is a variation of the standard PCR technique that is commonly used to
quantify DNA or RNA in a sample.( it can measure the initial quantity of nucleic acid of
interest)
how?

6. HOW IT WORKS
In real-time PCR, the amount of DNA is measured after each cycle via
fluorescent dyes that yield increasing fluorescent signal in direct
proportion to the number of PCR product molecules (amplicons)
generated.
Data collected in the exponential phase of the reaction yield quantitative
information on the starting quantity of the amplification target.
Fluorescent dye used in realtime PCR attached to PCR primers or probes
that hybridize with PCR product during amplification. The change in
fluorescence over the course of the reaction is measured by scanning
capability of the machine. By plotting fluorescence against the cycle
number, the real-time PCR instrument generates an amplification plot that
represents the accumulation of product over the duration of the entire
PCR reaction

7. WHAT HAPPENED IN THE MACHINE

8. Initially, fluorescence remains at background levels, and increases in fluorescence are not
detectable even though product accumulates exponentially. Eventually, enough amplified
product accumulates to yield a detectable fluorescent signal. The cycle number at which
this occurs is called the
threshold cycle, or CT.
The CT of a reaction is determined mainly by the amount of template present at the start of
the amplification reaction.
If a large amount of template is present at the start of the reaction, relatively few
amplification cycles will be required to accumulate enough product to give a fluorescent
signal above background. Thus, the reaction will have a low, or early, CT.
If a small amount of template is present at the start of the reaction, more amplification
cycles will be
required for the fluorescent signal to rise above background. Thus, the reaction will have a

10. REAL TIME PCR TERMINOLOGY

11. Baseline
The baseline of the real-time PCR reaction refers to the signal level during the initial
cycles of PCR, usually cycles 3 to 15, in which there is little change in fluorescent
signal.
Threshold
The threshold of the real-time PCR reaction is the level of signal that reflects a
statistically significant increase over the calculated baseline signal It is set to
distinguish relevant amplification signal from the background. Usually, real-time
PCR instrument software automatically sets the threshold at 10 times the standard
deviation of the fluorescence value of the baseline. However, the positioning of the
threshold can be set at any point in the exponential phase of PCR.
Ct (threshold cycle)
The threshold cycle (Ct) is the cycle number at which the fluorescent signal of the
reaction crosses the threshold. The Ct is used to calculate the initial DNA copy
number.

12. Standard curve
A dilution series of known template concentrations that entered in the same
reaction run. Serve two functions
1- Determining the initial starting amount of the target template in
experimental samples
2- Assessing the reaction efficiency.

13. Melting curve (dissociation curve)
Is an option found in all real time PCR instruments. In this technique the
instrument after the reaction start to raise the mixture temperature gradually that
lead to denature the fluorescently ladled double stranded DNA (product) causing
reduction in the fluorescent signal until the temperature reach the melting
temperature leading to significant drop in the fluorescent signal.

15. Functions of melting curve
Post-amplification melting-curve analysis is a simple, straightforward way to
check real-time PCR reactions for primer-dimer artifacts and to ensure reaction
specificity.
gDNA contamination in an RNA sample
• Splice variants (if there is extra sequence between primers)
Because the melting temperature of nucleic acids is affected by length, GC content,
and the presence of base mismatches, among other factors, different PCR products
can often be distinguished by their melting characteristics. The characterization of
reaction products (e.g., primerdimers vs. amplicons) via melting curve analysis
reducesthe need for time-consuming gel electrophoresis.

16. EXAMPLES OF MELTING CURVE

19. STEPS OF REAL TIME PCR
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.
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.

20. THE CORRECT REAL TIME PCR ASSAY
The properties of optimized qPCR assay are
Linear standard curve (R2 > 0.980 or r > |–0.990|)
• High amplification efficiency (90–105%)
• Consistency across replicate reactions.
A powerful way to determine whether your qPCR assay is optimized is to run serial
dilutions of a template and use the results to generate a standard curve. Which gives the
following information
1- Correlation coefficient (R2)
measure of how well the data fit the standard curve. The R2 value reflects the linearity of
the standard curve. Ideally, R2 = 1, although 0.999 is generally the maximum value.
Linearity, in turn, gives a
measure of the variability across assay replicates and whether the amplification
efficiency is the same for different starting template copy numbers.
2- Slope
The slope of the log-linear phase of the amplification reaction is a measure of reaction
efficiency. To obtain accurate and reproducible results, reactions should have an

21. Efficiency

22. THE ACCEPTED RANGE OF EFFICIENCY
An efficiency close to 100% is the best indicator of a optimal ,
reproducible assay.
The accepted amplification efficiency is 90–105%.
Low reaction ( less than 90) efficiencies may be caused by poor
primer design or by suboptimal reaction conditions.
Higher Reaction efficiencies (greater than 105) may indicate
pipetting error in your serial dilutions or coamplification of
nonspecific products, such as primer-dimers.

23. EXPERIMENTAL DESIGN
The first step in designing a real-time PCR experiment is to decide on the best type of
assay for the experiment of interest.
Should a singleplex or multiplex assay be used?
What type of real-time chemistry should one employ?
Multiplexing: amplification of more than one target in a single reaction tube. it is
possible to amplify and quantify as many as five targets in a single tube.
singleplex: amplification of only one target(gene).

24. WHEN YOU SHOULD DO MULTIPLEXING
Your amount of starting material is limited ( your
samples very hard to obtain)
Reduction in reagent costs
Minimization of sample handling and associated
opportunities for laboratory contamination
Your samples number very large

25. SELECTING THE FLUORESCENT LABEL
The variety of fluorescent chemistries available can be categorized into two major
types:
1- DNA-binding dyes >>>>>>>>>>> (SYBR Green I and 2)
2- dye-labeled, sequence-specific oligonucleotide primers or probes >>>>>
 molecular beacons
TaqMan
Hybridization
Eclipse probes
Amplifluor
Scorpions,
LUX
BD
 QZyme
The most commonly used chemistries for real-time PCR are the DNA-binding dye
SYBR Green I and TaqMan hydrolysis probes

26. DNA-BINDING DYES (SYBR GREEN I)
SYBR Green I, which binds nonspecifically to double-stranded DNA (dsDNA). SYBR Green
I
exhibits little fluorescence when it is free in solution, but its fluorescence increases up to
1,000-fold when it binds dsDNA
Advantages:
 Cheap
 Easy to assay
Disadvantage
 Has no specificity bind to any
double stranded DNA
 Can not be used for multiplex assay

27. TaqMan probe

28. Advantage
high specificity
high signal-to-noise ratio
ability to perform multiplex reactions.
Disadvantages: are that the initial cost of the probe may be high

30. DESIGN AND OPTIMIZATION OF SYBR GREEN I
REACTIONSThe steps for developing a SYBR Green I assay are:
• Primer design and amplicon design
• Assay validation and optimization
Primer design concerations
1- Design primers with a GC content of 50–60%
2- Maintain a melting temperature (Tm) between 50ºC and 65ºC. We calculate Tm values
using the nearest-neighbor method with values of 50 mM for salt concentration and 300
nM for oligonucleotide concentration
3- Avoid secondary structure; adjust primer locations outside of the target sequence
secondary structure if required
4- avoid repeat (tttt aaaa ggggg)
5- Place Gs and Cs on ends of primers
6- Check sequence of forward and reverse primers to ensure no 3' complementarity
(avoid primer-dimer formation)

31. Amplicon concedrations
• Design amplicon to be 75–200 bp. Shorter amplicons are typically amplified with
higher efficiency. An amplicon should be at least 75 bp to easily distinguish it from any
primer-dimers that might form
• Avoid secondary structure if possible.

32. OPTIMIZATION BY STANDARD CURVE
The efficiency, reproducibility, and dynamic range of a SYBR Green I assay can be
determined by constructing a standard curve using serial dilutions of a known template
It is important to note that the range of template concentrations used for the standard
curve must encompass the entire range of template concentration of the test samples to
show that results from the test samples are within the linear dynamic range of the assay.
If the test samples give results outside of the range of the standard curves, one of the
following must be performed:
• Construct a wider standard curve covering the test sample concentrations and perform
analysis to ensure that the assay is linear in that new range
• If the test samples give a lower CT than the highest concentration of standards used in
the standard curve, repeat the assay using diluted test samples
• If the test samples give a higher CT than the lowest concentration of standards used in
the standard curve, repeat the assay using larger amounts of the test samples

33. DESIGN AND OPTIMIZATION OF TAQMAN PROBE REACTIONS
A TaqMan assay uses:
a pair of PCR primers
a dual-labeled target-specific fluorescent probe.
Probe designation considerations
The TaqMan probe should have a Tm 5–10°C higher than that of the primers.
the probe should be <30 nucleotides
must not contain a G at its 5' end because this could quench the fluorescent signal even
after hydrolysis.
Choose a sequence within the target that has a GC content of 30–80%.
design the probe to anneal to the strand that has more Gs than Cs (so the probe
contains more Cs than Gs).