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’ 3’ d. NTPs Thermal Stable DNA Polymerase Primers 5’ 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’ Taq 3’ 5’ 3’ Taq 5’ Repeat

5’3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 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’ 5’ 3’ 5’ 3’ 5’3’ 5’ 3’

2*10=1024 4*5=1024 2*10=1024 4*5=1024

Ct values are directly related to the starting quantity of DNA, by way of the formula: Quantity = 2^ Ct Ct values are directly related to the starting quantity of DNA, by way of the formula: Quantity = 2^ Ct

Ct Values:

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 Excitation Wavelength 5’ 3’ 5’ 3’ 5’ Taq 3’ 5’ 3’ Taq 5’ Repeat ID

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

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., TrisCl, 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 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.  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.  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 Kit QuantiTect Reverse Transcription Kit Two step RT-PCR

3. Handling and storing primers

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 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 C T 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 C T 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

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, 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 T m s. Curves with peaks at a T m 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 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 T m s. Curves with peaks at a T m 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:quantitative PCRmedical laboratories Gene expressionGene expression assays Pharmacogenomics Human Leukocyte AntigenHuman Leukocyte Antigen (HLA) genotyping Determine the viral load in clinical specimens (HIV, Hepatitis)HIVHepatitis Bacterial Identification [7] assays [7] DNADNA quantification SNP genotyping Verification of microarray resultsmicroarray