•PCR reaction to be visualized “in real time” as the reaction progresses

•to quantify the amount of DNA in the sample at the start of the reaction

REAL TIME QUANTIFICATION

5’

5’

3’

3’

d.NTPs

Thermal Stable DNA Polymerase

Primers5’

3’5’

3’

5’

3’

5’

3’

5’

3’

5’

3’

5’

3’5’

3’

Denaturation

5’

3’

5’

3’

5’

3’

5’

3’

5’

3’5’

3’

Annealing

Add to Reaction Tube

THE REACTION

Extension

5’ 3’

5’3’5’ 3’

5’3’

Extension Continued

5’ 3’

5’3’5’

5’

Taq

Taq

3’

5’3’

Taq

Taq5’

5’

Repeat

5’3’5’ 3’

5’3’5’

3’

5’5’3’

3’

5’3’5’ 3’

Cycle 2

4 Copies

Cycle 3

8 Copies

5’3’5’

3’

5’3’5’ 3’

5’3’5’ 3’

5’3’5’ 3’

5’3’5’ 3’

5’3’3’5’

5’3’5’ 3’

5’3’5’ 3’

0

500000

1000000

1500000

2000000

2500000

3000000

3500000

4000000

4500000

5000000

0 5 10 15 20 25 30 35 40

2*10=1024

4*5=1024

0

500000

1000000

1500000

2000000

2500000

3000000

3500000

4000000

4500000

5000000

0 5 10 15 20 25 30 35 40

Ct values are directly related to the starting quantity of DNA, by way of the formula:

Quantity = 2^Ct

0

500000

1000000

1500000

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5000000

0 5 10 15 20 25 30 35 40

23 2528

Ct Values:

Copy Number vs. Ct - Standard Curve

y = -3.3192x + 39.772

R2 = 0.9967

0

5

10

15

20

25

30

35

40

0 1 2 3 4 5 6 7 8 9 10 11

Log of copy number (10n)

Ct

As shown by Higuchi et al.2, a plot of the log of initial target copy number for a set of standards versus CT is a straight line.

Effect of Limiting Reagents

CT is a more reliable measure of starting copy number than an endpoint measurement

the rate of target amplification decreases until a plateau is reached

1. double stranded DNA binding dye: SYBR GREEN ®

2. fluorogenic probes: Taqman ® Probes

Detection of PCR product accumulation

SYBR Green real time

Extension

5’ 3’

5’3’5’ 3’

5’3’

Apply ExcitationWavelength

5’ 3’

5’3’5’

5’

Taq

Taq

3’

5’3’

Taq

Taq5’

5’

Repeat

ID ID

ID IDID

ID ID ID

ID ID

l l l

ll

5700 system: able to use an internal reference dye (ROX)

normalize for non-PCR-related, well-to-well fluctuations in fluorescence

INTERNAL REFERENCE DYE

fluorescence readings taken at 95 ºC in the baseline region and is

essential for reproducible results.

Fluorophores: 6-carboxyfluorescein, acronym: FAM,

tetrachlorofluorescein, acronym: TET

Quenchers: tetramethylrhodamine, acronym: TAMRA,

 minor groove binder, MGB

fluorophore covalently attached to the 5’-end of the oligonucleotide probe and

a quencher at the 3’-end.

PROBE BASED real time

PROBE BASED real time

the 5' to 3' exonuclease activity of the polymerase degrades the probe that has annealed to the template.

Degradation of the probe releases the fluorophore from it and breaks the close proximity to the quencher, thus relieving the quenching effect and allowing fluorescence of the fluorophore.

Hence, fluorescence detected in the quantitative PCR thermal cycler is directly proportional to the fluorophore released and the amount of DNA

1. Template preparation and quality

2. Determining concentration and purity of nucleic acids

• Purity of nucleic acid templates is particularly important for real-time PCR, since

contaminants can interfere with fluorescence detection. more sensitive to impurities

such as proteins, phenol/chloroform, salts, and EDTA

• removal of genomic DNA

3. Storage of DNA and RNA

•kits, no degradation is detectable for at least 1 year

•Purified RNA should be stored at -20°C or -70°C in RNase-free water, under slightly

basic conditions (e.g., Tris•Cl, pH 8.0) acidic conditions can cause hydrolysis of DNA.

•Diluted solutions of nucleic acids (e.g., dilution series used as standards) should be

stored in aliquots and thawed once only.

•storage of aliquots in siliconized tubes where possible. This avoids adsorption of

nucleic acids to the tube walls, which would reduce the concentration of nucleic acids

in solution. For long-term storage of DNA samples, QIAsafe DNA Tubes and 96-Well

Plates, which provide stable, room-temperature storage.

4. RT-PCR

1. Assay design

A. Automated

B. Self designed

One-step RT-PCR combines the first-strand cDNA synthesis (reverse transcription) reaction

and PCR reaction in the same tube,

easier processing of large numbers of samples,

and helps minimize carryover contamination, since tubes are not opened between cDNA

synthesis and amplification. By amplifying the entire cDNA sample, one-step RT-PCR can

provide greater sensitivity—from as little as 0.01 pg total RNA.

One-step reactions allow for the use of sequence-specific primers only.

Two-step PCR begins with the reverse transcription of either total RNA or poly(A)+

RNA into cDNA using a reverse transcriptase.

When performing two-step RT-PCR, you have the option of using either oligo(dT),

random hexamer, or gene-specific primers,

then PCR is performed with either Platinum® Taq DNA Polymerase, Platinum® Taq

DNA Polymerase High Fidelity, or your choice of PCR enzyme.

2. One step vs two step RT-PCT

2. One step RT-PCR

Component

gDNA Wipeout Buffer

Quantiscript Reverse Transcriptase

Quantiscript RT Buffer

RT Primer Mix

Component

HotStarTaq DNA Polymerase

QuantiTect SYBR Green RT-PCR Buffer

dNTP mix

SYBR Green I dye

ROX dye

Omniscript and Sensiscript Reverse Transcriptases

QuantiTect SYBR® Green RT-PCR KitQuantiTect Reverse Transcription Kit

Two step RT-PCR

3. Handling and storing primers

Storage

buffer

Lyophilized primers should be dissolved in a small volume of low-salt buffer to

give a concentrated stock solution (e.g., 100 uM). We recommend using TE (10

mM Tris•Cl, 1 mM EDTA, pH 8.0) for standard primers.

Storage Primers should be stored in TE in small aliquots at -20°C. Standard primers are

stable under these conditions for at least 1 year. Repeated freeze-thaw cycles

should be avoided, since they may lead to degradation.

Dissolving

primers

Before opening a tube containing lyophilized primer, spin the tube briefly to

collect all material at the bottom of the tube. To dissolve the primer, add the

required volume of TE, mix, and leave for 20 minutes to allow the primer to

completely dissolve. Mix again and determine the concentration by

spectrophotometry as described below.

We do not recommend dissolving primers in water. They are less stable in water

than in TE and some may not dissolve easily in water.

Primer

quality

Select company carefully

1. No template control (NTC)

All quantification experiments should include an NTC, containing all the

components of the reaction except for the template. This enables detection of

contamination.

2. No RT control

All RT-PCR experiments should include a negative control to test for

contaminating DNA.

3. Positive control

CONTROLS

ABSOLUTE AND RELATIVE QUANTIFICATION

A standard curve (plot of CT value/crossing point against log of amount of

standard) is generated using different dilutions of the standard. The target and

each of the standards are amplified in separate tubes. The CT value of the target is

compared with the standard curve, allowing calculation of the initial amount of

the target. It is important to select an appropriate standard for the type of

nucleic acid to be quantified.

Absolute quantification

Absolute quantification

Relative quantification

With this method, the amounts of the

target genes ....

and the reference gene......house keeping gene

The expression level of the reference gene must not vary under different experimental conditions, or in

different states of the same tissue (e.g., "disease" versus "normal" samples).

The quantification procedure differs depending on whether the target genes and the reference gene are

amplified with comparable or different efficiencies. For determination of PCR efficiency,

The typical method to analyze real-time PCR data is the ΔΔCt method. Its mathematics assumes that the real-time PCR assay has a 100% amplification efficiency. As the primers deviate from this ideal, the error in the fold difference increases exponentially. The traditional method for determining amplification efficiency requires a calibration curve. Serially dilute an artificial template of known concentration. Plot the Ct values versus the initial amounts of input material on a semi-log10 plot, fit the data to a straight line (Fig) and calculate the slope. The closer the slope is to -3.33, the closer the amplification efficiency is to the 100% ideal.

MELTING CURVES

To carry out melting curve analysis, the temperature is increased very slowly from a low

temperature (e.g., 65°C) to a high temperature (e.g., 95°C). At low temperatures, all PCR

products are double stranded, so SYBR Green I binds to them and fluorescence is high, whereas

at high temperatures, PCR products are denatured, resulting in rapid decreases in fluorescence.

The fluorescence is measured continuously as the temperature is increased and plotted against

temperature. A curve is produced, because fluorescence decreases slightly through the lower end

of the temperature range, but decreases much more rapidly at higher temperatures as the

melting temperatures of nonspecific and specific PCR products are reached. The detection

systems calculate the first derivatives of the curves, resulting in curves with peaks at the

respective Tms. Curves with peaks at a Tm lower than that of the specific PCR product indicate the

formation of primer-dimers, while diverse peaks with different Tms or plateaus indicate production

of nonspecific products or a smear

Real-Time PCR has become a cornerstone of molecular biology:

• Gene expression analysis– Cancer research– Drug research

• Disease diagnosis– Viral quantification

• Food testing– Percent GMO food

• Transgenic research– Gene copy number

TaqMan probe-based assays are widely used in quantitative PCR in research and medical laboratories:Gene expression assaysPharmacogenomicsHuman Leukocyte Antigen (HLA) genotypingDetermine the viral load in clinical specimens (HIV, Hepatitis)Bacterial Identification[7] assaysDNA quantificationSNP genotypingVerification of microarray results