Protocol

@ OmicsGen LifeSciences Pvt Ltd

@ OmicsGen LifeSciences Pvt Ltd

OG/T7/2022/23

Contact us… Registered Address:

R&D Operations @ OmicsGen LifeSciences Pvt Ltd
OmicsGen LifeSciences Pvt Ltd V1/326B, Pallilankara, HMT Colony PO
Veegaland - K Chittilappilly Tower Kalamassery, Ernakulam,
Second Floor 683503
Opp. NPOL Main Gate
NGO Quarters - Bharata Mata College Road
Thrikkakara P.O, Kakkanad
Kochi - 682021

Phone: +919446971288
Email us: info@omicsgen.com

www.omicsgen.com
 RNA ISOLATION
Precautions to be taken while handling RNA:
Ribonucleases (RNases) are very stable and active enzymes that generally do not require
cofactors to function. Since RNases are difficult to inactivate and even minute amounts are
sufficient to destroy RNA. Do not use any plastic ware or glassware without first eliminating
possible RNase contamination. Great care should be taken to avoid inadvertently introducing
RNases into the RNA sample during or after the isolation procedure. In order to create and
maintain an RNase-free environment, the following precautions must be taken during
pretreatment while working with RNA.
 Always wear latex or vinyl gloves while handling reagents and RNA samples to prevent
RNase contamination from surface of the skin or form dusty laboratory equipment.
Change gloves frequently and keep tubes closed whenever possible.
 Collection tubes, tips, pipettes, electrophoresis unit etc. to be used for the experiment
must be UV treated for 15-20 minutes.
 Use sterile, disposable plastic ware and micropipettes reserved for RNA work to prevent
cross contamination with RNases from shared equipments.
 Non-disposable plastic wares should be treated before use to ensure that it is RNase-free.
Plastic ware should be thoroughly rinsed with 0.1M NaOH, 1mM EDTA followed by
RNase-free water. Alternatively, chloroform-resistant plastic ware can be rinsed with
chloroform to inactivate RNases.
 Glassware used for RNA work should be cleaned with detergent, thoroughly rinsed, and
oven baked at 24˚C for at least 4 hours before use. Alternatively glassware can be treated
with DEPC (diethyl pyrocarbonate). Fill glassware with 0.1% DEPC (0.1% in water),
allow to stand overnight at 37˚C, and then autoclave or heat to 100˚C for 15 minutes to
eliminate residual DEPC.
 Electrophoresis tanks should be cleaned with detergent solution (e.g., 0.5% SDS),
thoroughly rinsed with RNase-free water, and then rinsed with ethanol and allowed to
dry.
 Solutions (water and other solutions) should be treated with 0.1% DEPC.
Total RNA Extraction procedure is designed for rapid purification of RNA from different
samples.

Procedure
1. Grind the sample in motor and pestle with liquid nitrogen and 1ml of Trizol
2. Add 200µl of Chloroform to it.
3. Centrifuge at 12000 rpm for 10 minutes at 4oC
4. Collect the aqueous part and add 500µl of Isopropanol
5. Incubate at -20oC for 30 minutes
6. Centrifuge at 12000 rpm for 10 minutes at 4oC
7. Discard the supernatant and add 75% ethanol
8. Centrifuge at 12000 rpm for 10 minutes at 4oC
9. Discard ethanol and collect the pellet
10. Resuspend the pellet in 30µl of nuclease free water
 cDNA SYNTHESIS

Thermo Scientific RevertAid First Strand cDNA Synthesis Kit

DESCRIPTION

The Thermo Scientific™ RevertAid™ First Strand cDNA Synthesis Kit is a complete system for
efficient synthesis of first strand cDNA from mRNA or total RNA templates. The kit uses
RevertAid Reverse Transcriptase (RT), which has lower RNase H activity compared to AMV
reverse transcriptase. The enzyme maintains activity at 42-50°C and is suitable for synthesis of
cDNA up to 13 kb.

The recombinant Thermo Scientific™ RiboLock™ RNase Inhibitor, supplied with the kit,
effectively protects RNA from degradation at temperatures up to 55°C. First strand cDNA
synthesized with this system can be directly used as a template in PCR or real-time PCR. It is
also ideal for second strand cDNA synthesis or linear RNA amplification. Radioactively and
non-radioactively labeled nucleotides can be incorporated into first strand cDNA for use as a
probe in hybridization experiments, including microarrays.

STORAGE

All components of the kit should be stored at -20°C. Keep control RNA at -70°C for longer
storage.

IMPORTANT NOTES

Avoiding ribonuclease contamination RNA purity and integrity are essential for synthesis of full
length cDNA. RNA can be degraded by RNase A, which is a highly stable contaminant found in
any laboratory environment. General recommendations to avoid RNase contamination:

• DEPC-treat all tubes and pipette tips to be used in cDNA synthesis or use certified nuclease-
free lab ware.
• Wear gloves when handling RNA and all reagents, as skin is a common source of RNases.
change gloves frequently.

• Use RNase-free reagents, including high quality water (e.g., Water, nuclease-free, #R0581).

• Use RiboLock™ RNase Inhibitor (provided with the kit) to protect RNA from the activity of
RNases.

• Keep all kit components tightly sealed when not in use. Keep all tubes tightly closed during the
reverse transcription reaction.

Template RNA

Total cellular RNA isolated by standard methods is suitable for use with the kit. Purified RNA
must be free of salts, metal ions, ethanol and phenol to avoid inhibiting the cDNA synthesis
reaction. Trace contaminants can be removed by ethanol precipitation of the RNA followed by
two washes of the pellet with cold 75% ethanol. For RT-PCR applications, template RNA must
be free of DNA contamination. Prior to cDNA synthesis, RNA can be treated with DNase I,
RNase-free (#EN0521) to remove trace amounts of DNA. Always perform a control reaction
which includes all components for RTPCR except for the reverse transcriptase enzyme.
Removal of genomic DNA from RNA preparations

1. Add to an RNase-free tube:

RNA =1 µg
10X Reaction Buffer with MgCl2 =1 µL
DNase I, RNase-free =1µL (1 U)
Water, nuclease-free= to 10 µL
* Do not use more than 1 U of DNase I, RNase-free per 1 µg of RNA.

2. Incubate at 37°C for 30 min.

3. Add 1 µl 50 mM EDTA and incubate at 65°C for 10 min. RNA hydrolyzes during heating
with divalent cations in the absence of a chelating agent (1). Alternatively, use
phenol/chloroform extraction.

4. Use the prepared RNA as a template for reverse transcriptase.

RNA sample quality

Assess RNA integrity prior to cDNA synthesis. The most common method is denaturing agarose
gel electrophoresis followed by ethidium bromide staining. If both 18S and 28S rRNA appears as
sharp bands after electrophoresis of total eukaryotic RNA, the RNA is considered to be intact.
The 28S rRNA band should be approximately twice as intense as the 18S rRNA. Any smearing
of rRNA bands is an indication of degraded mRNA. If this occurs, a new sample of total RNA
should be prepared.

RNA quantity

• Use 0.1 ng - 5 µg of total RNA or 1 ng - 500 ng of poly (A) mRNA to generate first strand
cDNA as the initial step of a two-step RT-PCR protocol.

• Use 1 µg of isolated mRNA to generate first strand cDNA for second-strand synthesis and
subsequent cloning reactions.

PROTOCOLS

I. First Strand cDNA Synthesis After thawing, mix and briefly centrifuge the components of the
kit. Store on ice.

1. Add the following reagents into a sterile, nuclease- free tube on ice in the indicated order:

Template RNA total RNA or poly(A) mRNA 1. ng - 5 µg

or specific RNA 10 pg - 0.5 µg
0.01 pg - 0.5 µg

Primer Oligo (dT)18 primer or 0.5µL

Random Hexamer primer or 0.5µL
 gene-specific primer 7.5-15 mol

to 6 µL
Water, nuclease-free

Total volume 6 µL

2. Optional. If the RNA template is GC-rich or contains secondary structures, mix gently,
centrifuge briefly and incubate at 65°C for 5 min. Chill on ice, spin down and place the
vial back on ice.

3. Add the following components in the indicated order:

5X Reaction Buffer 2µL

RiboLock RNase Inhibitor (20 0.5 µL

U/µL)

10 mM dNTP Mix 1 µL

RevertAid M-MuLV RT 0.5µL

(200U/µL)

4. Mix gently and centrifuge briefly.

5. For oligo (dT) 18 or gene-specific primed cDNA synthesis, incubate for 60 min at
42°C. For random hexamer primed synthesis, incubate for 5 min at 25°C followed by 60
min at 42°C. Note. For GC-rich RNA templates the reaction temperature can be increased
up to 45°C.
6. Terminate the reaction by heating at 70°C for 5 min.
The reverse transcription reaction product can be directly used in PCR applications or
stored at -20°C for less than one week. For longer storage, -70°C is recommended.
 II. PCR Amplification of First Strand cDNA
The product of the first strand cDNA synthesis can be used directly in PCR or qPCR.
The volume of first strand cDNA synthesis reaction mixture should not comprise more
than 1/10 of the total PCR reaction volume. Normally, 2 µL of the first strand cDNA
synthesis reaction mixture is used as template for subsequent PCR in 50 µL total volume.

CONTROL REACTIONS
Positive and negative control reactions should be used to verify the results of the first
strand cDNA synthesis steps.
• Reverse transcriptase minus (RT-) negative control is important in RT-PCR or RT-
qPCR reactions to assess for genomic DNA contamination of the RNA sample. The
control RT- reaction contains every reagent for the reverse transcription reaction except
for the RT enzyme.
• No template negative control (NTC) is important to assess for reagent contamination.
The NTC reaction contains every reagent for the reverse transcription reaction except for
RNA template.
• Positive control RNA template and gene-specific primers are supplied with the kit. The
human GAPDH control RNA (1.3 kb) was produced by in vitro transcription. The
GAPDH-specific control PCR primers are designed to be complementary to human,
mouse and rat GAPDH genes and generate 496 bp RT-PCR product. The protocol for the
positive control RT-PCR is provided below.
I. Control first strand cDNA synthesis reaction

Mix and briefly centrifuge all components after thawing, keep on ice.
1. Add the following reagents into a sterile, nuclease- free tube on ice in the indicated order:

Control GAPDH RNA (50 ng/µL) 1 µL

Oligo (dT)18 Primer or Random Hexamer Primer or Reverse 0.5µL

GAPDH Primer
 5X Reaction Buffer 2µL

RiboLock RNase Inhibitor (20 U/µL) 0.5 µL

10 mM dNTP Mix 1 µL

RevertAid RT (200 U/µL) 0.5µL

Water, nuclease-free 4.5 µL

Total volume 10 µL

2. Mix gently and centrifuge.

3. For oligo (dT) 18 or gene-specific primed cDNA synthesis, incubate for 60 min at 42°C. For
random hexamer primed synthesis, incubate for 5 min at 25°C followed by 60 min at 42°C.

4. Terminate the reaction by heating at 70°C for 5 min.

5. Briefly centrifuge and precede with control PCR amplification.

II. Control PCR amplification

1. Dilute the cDNA generated with the control first strand cDNA reaction 1:1000 in Water,
nuclease free.

2. Gently vortex and briefly centrifuge all PCR reagents after thawing.

3. Place a thin-walled PCR tube on ice and add the following reagents:

cDNA from control RT reaction 1µL

(1:1000dilution)

10X PCR buffer 2.5 µL

10 mM dNTP Mix 0.5 µL (0.2 mM

each)
 25 mM MgCl2 1.5µL

Forward GAPDH Primer 0.75 µL

Reverse GAPDH Primer 0.75 µL

Taq DNA polymerase (5 U/µL) 0.25 µL

Water, nuclease-free 17.75 µL

Total volume 25 µL

4. Perform PCR in a thermal cycler with a heated lid or overlay with 25 µL of mineral oil.

Step Temperature Time Number of

°C cycles

Initial denaturation 94 3 min 1

Denaturation 94 30 sec 35

Annealing 58 30 sec 35

Extension 72 45 se 35
c

5. Load 5-10 µl of the RT-PCR product on 1% agarose gel.

6. A distinct 496 bp PCR product should be visible after ethidium bromide staining.
 Real Time PCR

As the name suggests, real time PCR is a technique used to monitor the progress of a PCR
reaction in real time. At the same time, a relatively small amount of PCR product (DNA, cDNA
or RNA) can be quantified. Real Time PCR is based on the detection of the fluorescence
produced by a reporter molecule which increases, as the reaction proceeds. This occurs due to
the accumulation of the PCR product with each cycle of amplification. These fluorescent reporter
molecules include dyes that bind to the double-stranded DNA (i.e. SYBR® Green ) or sequence
specific probes (i.e. Molecular Beacons or TaqMan® Probes). Real time PCR facilitates the
monitoring of the reaction as it progresses. One can start with minimal amounts of nucleic acid
and quantify the end product accurately. Moreover, there is no need for the post PCR processing
which saves the resources and the time. These advantages of the fluorescence based real time
PCR technique have completely revolutionized the approach to PCR-based quantification of
DNA and RNA. Real time PCR assays are now easy to perform, have high sensitivity, more
specificity, and provide scope for automation. Real time PCR is also referred to as real time RT
PCR which has the additional cycle of reverse transcription that leads to formation of a DNA
molecule from a RNA molecule. This is done because RNA is less stable as compared to DNA
Real Time PCR procedure
In a real time PCR protocol, a fluorescent reporter molecule is used to monitor the PCR as it
progresses. The fluorescence emitted by the reporter molecule manifolds as the PCR product
accumulates with each cycle of amplification. Based on the molecule used for the detection, the
real time PCR techniques can be categorically placed under two heads:
1. Non-specific Detection using DNA Binding Dyes.
2. Specific Detection Target Specific Probes.

Non-specific Detection using DNA Binding Dyes

In real time PCR, DNA binding dyes are used as fluorescent reporters to monitor the real time
PCR reaction. The fluorescence of the reporter dye increases as the product accumulates with
each successive cycle of amplification. By recording the amount of fluorescence emission at
each cycle, it is possible to monitor the PCR reaction during exponential phase. If a graph is
drawn between the log of the starting amount of template and the corresponding increase the
fluorescence of the reporter dye fluorescence during real time PCR, a linear relationship is
observed.
SYBR® Green is the most widely used double-strand DNA-specific dye reported for real time
PCR. SYBR® Green binds to the minor groove of the DNA double helix. In the solution , the
unbound dye exhibits very little fluorescence. This fluorescence is substantially enhanced when
the dye is bound to double stranded DNA. SYBR® Green remains stable under PCR conditions
and the optical filter of the thermocycler can be affixed to harmonize the excitation and emission
wavelengths. Ethidium bromide can also be used for detection but its carcinogenic nature renders
its use restrictive.
Although these double-stranded DNA-binding dyes provide the simplest and cheapest option for
real time PCR, the principal drawback to intercalation based detection of PCR product
accumulation is that both specific and nonspecific products generate signal.
Specific Detection using Target Specific Probes
Specific detection of real time PCR is done with some oligonucleotide probes labeled with both a
reporter fluorescent dye and a quencher dye. Probes based on different chemistries are available
for real time detection, these include:
a. Molecular Beacons
b. TaqMan® Probes
c. FRET Hybridization Probes
d. Scorpion® Primers

Real Time PCR Applications Include

1 . Quantitative mRNA expression studies.
2 . DNA copy number measurements in genomic or viral DNAs.
3 . Allelic discrimination assays or SNP genotyping.
4 . Verification of microarray results.
5 . Drug therapy efficacy.
6 . DNA damage measurement.
Real Time PCR VS Traditional PCR
Real time PCR allows for the detection of PCR product during the early phases of the reaction.
This ability of measuring the reaction kinetics in the early phases of PCR provide a distinct
advantage over traditional PCR detection. Traditional methods use gel electrophoresis for the
detection of PCR amplification in the final phase or at end-point of the PCR reaction.

Limitations of End-point PCR

In a PCR reaction as the reaction progresses, the reagents are being consumed as a result of
amplification. Now the PCR product is no longer being doubled at each cycle due to this reagent
constraint. This depletion will occur at different rates for each replicate. Thus, the samples begin
to diverge in their quantities. This diminished amplification is the linear phase of the reaction.
The plateau for each tube will differ due to the different reaction kinetics for each sample. It is in
this phase where traditional PCR takes its measurement, also known as the end-point. This End-
Point Detection has some problems such as low resolution, poor precision, low sensitivity and
the need for post PCR processing. Also, the results of this detection are not expressed in numbers
and there is no scope for automation.
Real Time PCR Primer and Probe Design with AlleleID® & Beacon Designer™
Beacon Designer™ is a comprehensive real time PCR primer and probe design tool for
designing single template and multiplex assays. Real time PCR chemistries supported include
Molecular beacons, TaqMan®, FRET, Scorpions® and SYBR® Green. It designs molecular
beacons for standard and NASBA® assays and designs LNA spiked Taqman® probes as well.
AlleleID® supports all the major features of Beacon Designer™ except NASBA® assay, LNA™
spiked Taqman® probe design and multiplex assay design. TaqMan® MGB probe design is
unique to AlleleID® (whereas regular TaqMan® probes based assays can be designed using both
AlleleID® and Beacon Designer™. AlleleID® is written for advance real time PCR applications
such as oligo design for species identification and taxa discrimination. It includes a multiple
sequence alignment module for identifying conserved and species specific regions. AlleleID®
also includes support for real time PCR primer design over exon junctions for selective
amplification of cDNA.
Real Time PCR setting
Reaction Volume - 6ul
2X Real Time PCR smart mix- 3ul
Forward primer+Reverse primer-0.5ul
Template cDNA - 1ul
ddNFW - 1.5ul

PCR Program
Polymerase activation
95 °C 10 minutes

Denaturation
95 °C 15 sec
40 cycles
Annealing/extension
60 °C 1 minute

Real-time PCR, also called quantitative PCR or qPCR, can provide a simple and elegant method
for determining the amount of a target sequence or gene that is present in a sample. Its very
simplicity can sometimes lead to problems by overlooking some of the critical factors that make
it work. This review will highlight these factors that must be considered when setting up and
evaluating a real-time PCR reaction.
Figure 1. Graphical representation of real-time PCR data. Rn is the fluorescence of the reporter
dye divided by the fluorescence of a passive reference dye; i.e.,Rn is the reporter signal
normalized to the fluorescence signal of Applied Biosystems™ ROX™ Dye. (A) In this view,
Rn is plotted against PCR cycle number. (B) ΔRn is Rn minus the baseline; ΔRn is plotted
against PCR cycle number. (C) An amplification plot shows the variation of log (ΔRn) with PCR
cycle number.

Factors that can influence Ct

Ct (threshold cycle) is the intersection between an amplification curve and a threshold line
(Figure 1B). It is a relative measure of the concentration of target in the PCR reaction. Many
factors impact the absolute value of Ct besides the concentration of the target. We will discuss
the most common template-independent factors that can influence Ct and describe how to
evaluate the performance of a real-time PCR reaction.
Figure 1, above, shows several parameters of the real-time reaction amplification plot. The
exponential phase in Figure 1B corresponds to the linear phase in Figure 1C. The threshold must
be set in the linear phase of the amplification plot in Figure 1C. The Ct value increases with a
decreasing amount of template. However, artifacts from the reaction mix or instrument that
change the fluorescence measurements associated with the Ct calculation will result in template-
independent changes to the Ct value. Therefore, the Ct values from PCR reactions run under
different conditions or with different reagents cannot be compared directly.

Figure 2. Raw fluorescence data obtained from one assay using two master mixes with the same
ROX level. The difference in signal is due to the master mix composition. The reaction was
performed on an Applied Biosystems™ 7900HT Fast Real-Time PCR System using a Applied
Biosystems™ VIC™/MGB probe. The x-axis shows the emission wavelength of the fluorophore
and the y-axis shows the intensity of the emission.
The effect of master mix components
The fluorescence emission of any molecule is dependent on environmental factors such as the pH
of a solution and salt concentration. Figure 2 shows the raw fluorescence data of an Applied
Biosystems™ TaqMan® probe in the background of two different master mixes. Note that the
fluorescence intensity is higher in Master Mix even though the target, probe, and ROX dye
concentrations are the same in both cases.

The resulting ΔRn value will, therefore, vary as shown in Figure 3. Note that the baseline
fluorescence signals, in a template-independent factor, are different for the two master mixes
(Figure 3A). Variations in the Ct value do not reflect the overall performance of the reaction
system (Figure 3B). Master mixes with equivalent sensitivity may have different absolute Ct
values.
Figure 3. Amplification of RNase P in equal amounts of human gDNA using Master Mix A and
Master Mix B. (A) Rn is plotted against cycle number and the baselines for both reactions are
shown. (B) log (ΔRn) is plotted against cycle number. The threshold (green line) is set at the
same level for both master mixes. The Ct value of Master Mix B (CtB) is earlier than that of
Master Mix A (CtA) for identical concentrations of target, reflecting the lower baseline of
Master Mix B. All amplifications were performed using the Applied Biosystems™ 7500 Real-
Time PCR System.

ROX passive reference dye

The Rn value is calculated as the ratio of the fluorescence of Applied Biosystems™ FAM™ Dye
divided by the fluorescence of ROX dye. Therefore, a lower amount of ROX dye would produce
a higher Rn value assuming fluorescence signal from FAM dye is unchanged. This would lead to
an increase in baseline Rn and subsequently a smaller ΔRn as well as a different Ct value. The
new Ct value obtained by lowering the level of ROX dye has no bearing on the true sensitivity of
the reaction, but can have other unintended consequences. Low concentrations of ROX dye can
result in increased standard deviation of the Ct value, as shown in Figure 4. The greater the
standard deviation, the lower the confidence in distinguishing between small differences in target
concentration (see the precision section below).
Figure 4. Amplification of TGF-beta using master mixes containing 3 different concentrations
of ROX dye. The variation of (A) Ct value and (B) standard deviation with ROX dye
concentration is shown. Decreasing the ROX dye concentration gives an earlier Ct, but increases
the standard deviation. All amplifications were performed using the Applied Biosystems 7500
Real-Time PCR System.

Efficiency of a PCR reaction

The efficiency of a PCR reaction can also affect Ct. A dilution series amplified under low
efficiency conditions could yield a standard curve with a different slope from one amplified
under high efficiency conditions. In Figure 5, two samples (X and Y) amplified under low and
high efficiency conditions show different Ct values for the same target concentration. In this
example, although the high-efficiency condition (the blue curve in Figure 5) gives a later Ct at
high concentrations, it results in better sensitivity at low target concentrations. The PCR
efficiency is dependent on the assay, the master mix performance, and sample quality. Generally,
efficiency between 90 and 110% is considered acceptable. the other, could be valuable in
concluding that there is less template in the first sample, assuming all other factors such as
instruments, reagents, and assays are equal. However, this is not true if different instruments,
reagents, primers and probes, or reaction volumes are involved in producing the two Ct's.
Therefore, the absolute Ct value comparison is only meaningful when comparing experiments
using the same reaction conditions as defined above.
Figure 5. Variation of Ct with PCR efficiency. The blue standard curve has an efficiency of
100% (the slope is –3.3). The green standard curve has an efficiency of 78% (the slope is –4).
Amplification of quantity Y gives an earlier Ct under low efficiency conditions (green)
compared to the high efficiency condition (blue). With a lower quantity (X) there is an inversion
and the low efficiency condition (green) gives a later Ct than the high efficiency condition (blue).

How to evaluate the performance of a real-time PCR reaction

In order to compare two reactions where a condition is changed (for example two different
master mixes or two different instruments), the following parameters must be evaluated.
Dynamic range
To properly evaluate PCR efficiency, a minimum of 3 replicates and a minimum of 5 logs of
template concentration are necessary. The reason for this suggested level of rigor is illustrated in
Figure 6, which demonstrates the possible mathematical variation of slope or efficiency obtained
when testing dilutions over 1 log vs. 5 logs. Thus, even if the assay is 100% efficient, a range
from 70 to 170% can be obtained when testing a dilution series of a single log, due to the
standard deviation in one dilution. For the same number of dilutions or replicates on a 5-log
range, the potential artifact is only ±8 %. That means for 94% efficiency on a 5-log range, the
assay would have a range of 88% to 100% efficiency. To accurately determine the efficiency of a
PCR reaction, a 5-log dilution series must be performed. A slope of –3.3 ±10% reflects an
efficiency of 100% ±10%. A PCR reaction with lower efficiency will have lower sensitivity.
R2 value
Another critical parameter to evaluating PCR efficiency is R2, which is a statistical term that
indicates how good one value is at predicting another. When R2 is 1, the value of Y (Ct) can be
used to accurately predict the value of (Figure 7A). If R2 is 0, the value of X cannot be
predicted from the value of Y (Figure 7B). An R2 value >0.99 provides good confidence in
correlating two values.
Precision
The standard deviation (square root of the variance) is the most common measure of precision. If
many data points are close to the mean, the standard deviation is small; if many data points are
far from the mean, the standard deviation is large.
In practice, a data set with a sufficient number of replicates forms an approximately normal
distribution. This is frequently justified by the classic central limit theorem which states that
sums of many independent, identically distributed random variables tend towards the normal
distribution as a limit. As shown in Figure 8A, approximately 68% of the values are within 1
standard deviation of the mean, 95% are within two standard deviations, and 99.7% lie within 3
standard deviations.
If a PCR is 100% efficient, the Ct difference between two successive concentrations in a 2-fold
dilution is 1 (Figure 8B). To be able to quantify a 2-fold dilution in more than 99.7% of cases,
the standard deviation has to be ≤0.167. The greater the standard deviation, the lower the ability
to distinguish between 2-fold dilutions. To be able to discriminate between a 2-fold dilution in
more than 95% of cases, the standard deviation has to be ≤0.250 (Figure 8C).
Sensitivity
Any system capable of effectively amplifying and detecting one copy of starting template has
achieved the ultimate level of sensitivity, regardless of the absolute value of the Ct.
As described earlier, efficiency is a key factor in determining the sensitivity of a reaction (Figure
5). Another important consideration when detecting very low copy numbers is that normal
distribution of template is not expected. Instead, a Poisson distribution is followed, which
predicts that in a large number of replicates containing an average of one copy of starting
template, approximately 37% should actually have no copies, only 37% should contain one copy,
and 18% should contain two copies (see Figure 9). Thus, for a reliable low copy detection, a
large number of replicates are necessary to provide statistical significance and to overcome the
Poisson distribution limitation.

Figure 6. Accurate calculation of PCR efficiency depends on the range of template amount used
for the dilution series. For a 2-fold dilution with 5 points (orange), the potential artifact is higher
than for the 10-fold dilution with 5 points (blue).
Figure 7. Examples of R2 values calculated for 2 straight lines. (A) There is a direct relation
between x and y values. (B) There is no relation between x and y values.

Figure 8. Normal distribution and standard deviation. (A) Normal distribution of data is shown.
For a PCR efficiency of 100%, the difference in Ct between the means of two successive
samples in a 2-fold dilution series is 1 (sample X and sample Y). (B) To be able to quantify both
samples in 99.7% of cases, the standard deviation has to be less than 1 Ct divided by 6 standard
deviations (1/6 = 0.167). (C) To be able to quantify both samples in 95% of cases, the standard
deviation has to be less than 1 Ct divided by 4 standard deviations (1/4 = 0.25).
Figure 9. Poisson distribution for low copy number. The blue curve represents Poisson
distribution for 3.3 pg of DNA (1 copy of DNA). The pink curve represents Poisson distribution
for 6.6 pg of DNA (1 cell, 2 copies of DNA).
Conclusion
Efficiency, R2, precision, and sensitivity are used to determine performance of a PCR reaction
when comparing different reaction conditions. For a rigorous evaluation, all factors listed in
Table 1 must be evaluated together. In addition to these factors, proper experimental controls
(such as no template control, no RT control) and template quality must be evaluated and
validated.
Analysis of gene expression data using qbase+.
• create an experiment in qbase+
• import run data
• customize sample properties
• select reference genes
• assess the quality of your qPCR data
• visualize and export the results
2. What is Relative Quantitation?
Methods for relative quantitation of gene expression allow you to quantify differences in the
expression level of a specific target (gene) between different samples. The data output is
expressed as a fold-change or a fold-difference of expression levels. For
example you might want to look at the change in expression of a particular gene over a given
time period in treated vs. untreated samples. For this hypothetical study, you can choose a
calibrator sample (i.e. untreated at day 0) and an endogenous control gene to normalize input
amounts. For all samples, levels of both target and endogenous control genes would be assessed
by real-time PCR. The results (target levels normalized to endogenous control levels) would then
be expressed in a format such as “At day 30, sample A had a 10-fold greater expression level of
the target gene than at day 0”.
 PRIMER DESIGNING
PCR is an essential and ubiquitous tool in genetics and molecular biology. For amplifying any
nucleotide sequence by PCR, two short stretches of DNA flanking the sequence to be amplified,
called the forward and reverse primers, need to be provided. Primer designing is important to
maintain specificity of PCR reactions. If the primer is not specific, non-specific amplicons will
be obtained.
Factors to be considered in primer designing
1. Primer should be 17-30 bases long(18-20 is optimum)
2. Specificity increases with increasing GC content. A GC content of 50% is ideal.
3. Primers with low GC content should be long enough to avoid low melting temperature.
4. Repeat sequences should be avoided ( >3-4 ).
5. Preferable to have G/C at the 3’ end.

Melting temperature of a PCR reaction,

Tm = 4 (GC) + 2 (AT),
Where the number of G or C and A or T are of the primers selected. The annealing
temperature,
Ta = Tm - 5ºC.

Primer Designing Software

There are many programs for primer design, commercial and free the following
workfine but so do many other programs. You don’t have to use these if you want to
use other programs.

Product Size Ranges : Enter the size range of the Amplicon you wish
Number to Return : number of primer alternative usually set to 5
or 10.
Max Tm Difference : difference in primer Tm set to 2
Primer GC% Min : set to 40 Opt: set to 50 Max : set to 60
Max Self Complementarity : set to 2 to start with may need to relax this if no
primers found i.e. increase value by 1*
Max 3' Self Complementarity : set to 0 to start with may need to relax this if no
primers found i.e. increase value by 1*
Max Poly-X : set to 3
*lower values more stringent.

These are basic guide to primer3 but provide a good starting point for primer design
but these values are not always ideal for every primer you need to design just most of
them. Reading the accompanying help pages etc. on the web site will give you a more
in depth guide to the options.

-The End-