Important Parameters of Quantitative PCR (qPCR) Analysis

Exponential Phase

It is important to quantitate your qPCR at the early part of the exponential phase of amplification instead at the later cycles or at the plateau. At the beginning of the exponential phase, all reagents are still in excess. This means the low amount of product will not compete with the primers’ annealing capabilities and the DNA polymerase is still highly efficient, making your data more accurate.

Threshold Ct

The threshold for Ct determination should be set up as close as possible to the base of the exponential phase. For precise comparison, compare Ct values from different experiments with the threshold defined in the same way.

Setting the Baseline

The baseline in real-time PCR should be set up carefully to allow accurate Ct determination. The baseline should be wide enough to eliminate the background found in early cycles of amplification, but should not overlap with the area in which the amplification signal begins to rise above background. For precise comparison, compare Ct values from different experiments with the baseline defined in the same way.

Efficiency of PCR (Slope)

Slope/Efficiency: Ideally the efficiency (E) of a PCR should be 100%, meaning that for each cycle the amount of product doubles (E=2).
This efficiency is calculated from the slope(s) of the standard curve according to the following formulas:
E = 10(-1/slope)-1
Log E = (-1/slope)log 10 – log 1
Log 2 = (-1/slope) x1 – 0 (because E=2, log 1= 0 and log10 = 1)
Slope= -1/log2 (after multiplying both sides by (slope/log2)
Slope = -3.32
For an efficiency of 100%, the slope is -3.32. A good reaction should have an efficiency between 90% and 110%, which corresponds to a slope between -3.58 and -3.10.

R2 Value

To evaluate the performance of a primer set, analyze a serial dilution of the target (10-fold dilution for example, over 5 to 7 log). The sample can be either a gene-specific plasmid or a cDNA preparation in which the gene of interest is known to be present. R2 is the coefficient of correlation obtained for the standard curve and should be >0.99.

Standard Curve

The analysis of the standard curve gives important information. To evaluate the performance of a primer set, analyze a serial dilution of the target (e.g. 10-fold dilution for over 5 to 7 log). The sample can be either a gene-specific plasmid or a cDNA preparation in which the gene of interest is known to be present.

Determination of Copy Number

How many copies are in a certain amount of human genomic DNA?
1 genome copy = 3 x 109 bp
1 bp = 618 g/mol
1 genome copy = (3 x 109 bp) x (618 g/mol/bp) = 1.85 x 1012 g/mol
= (1.85 x 1012 g/mol) x (1 mole/6.02 x 1023 (Avogadro’s number))
=3.08 x 1012 g
Each somatic cell has 6.16 pg of DNA (sperm and egg cells have 3.08 pg). There is one copy of every non-repeated sequence per 3.08 pg of human DNA. Therefore, 100 ng of genomic DNA would have:
(100,000 pg of DNA )/3.08 pg =~33 000 copies
1 ng of DNA has 330 copies
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PCR and Primer Design Considerations

Oligonucleotides are frequently used as primers in PCR. Unlike most large DNA, where the overall representation of the 4 bases averages out to about equal numbers of each, the base composition of an oligonucleotide (usually 15–30 bases) can vary greatly and affect the properties of the oligonucleotide. Key properties that are sequence dependent in oligonucleotides include extinction coefficient (1), A260/280 ratio (1), electrophoretic migration (2), and ethidium bromide staining (2). Use the free online OligoPerfect™ Designer software for primer design. Visit www.invitrogen.com and select OligoPerfect™ Designer from the Custom Primers menu.
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Tm Calculations

Wallace Method (for oligos <18mers): Tm = 2 x (A + T) + 4 x (G + C) 
%GC Method: Tm = 81.5 + 16.6 (log10[Na+] + 0.41[%GC] - [625/N]) 
N = length of oligo 
Nearest neighbor (1) Tm = (ΔH - 3.4 kcal)/((A + ΔS) + (R ln(C/4))) - 273.15 + 16.6 log10[salt] 
ΔH is the sum of nearest neighbor enthalpy changes 
A is the initiation constant of -10.8 cal/Ko mole for non-self complementary sequences, -12.4 cal/Ko mole for complementary sequences 
ΔS is the sum of nearest neighbor entropy changes 
R is the gas constant 1.987 cal/Ko mole 
C is the concentration of oligonucleotide (generally fixed at 250 pM) (2)

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PCR Optimization

PCR Optimization Guidelines
ADDITIVE Glycerol (5-10%), formamide (1-5%), or DMSO (2-10%) can be added to the PCR for template DNA with high GC content (these change the Tm of primer-template hybridization reaction and the thermostability of polymerase enzyme). Betain (0.5-2 M) is also useful in PCR of high GC content and long DNA. Perform a titration to determine optimum concentration. When using betain, reduce melting temperature (92-93°C) and annealing temperature (1-2°C lower). BSA (up to 0.8 ug/ul) may also improve efficiency of PCR reaction. PCRx Enhancer Solution (Cat. No. 11495-017) is a novel PCR cosolvent that facilitates efficient amplification of GC-rich sequences and remedies difficulties associated with PCR of problematic templates.
ANNEALINGUse appropriate temperatures based on the calculated Tm of primers.
CYCLING Denaturation time can be increased if template GC content is high. Extension time should be increased for larger PCR products, but this might lead to damage of the enzyme. The number of cycles may be increased if the amount of template DNA is very low, and decreased if template DNA is abundant.
DENATURING Use a temperature appropriate for polymerase of choice.
DNTP The concentrations of dNTPs used in a reaction are determined by the affinity of the enzyme for substrate (the Km of the enzyme). dNTPs on the order of 10uM are generally appropriate. Concentration of dNTPs decreases as the reaction proceeds. Excessive dNTP concentration reduces polymerase accuracy and requires higher Mg2+ in the reaction mix. Up to 1.5 mM dNTP may be used. dNTPs chelate Mg2+. Excessive dNTP concentration may increase the error rate. Lowering the dNTP (10-50 uM) may reduce error rate. Large PCR fragments require more dNTPs than small fragments.
EXTENSION Typically 72°C. At 70-72°C the activity is optimal, and primer extension occurs at up to 100 bases/sec. One minute is sufficient for reliable amplification of 2 kb sequences. Longer products require longer times: 1 minute per 1 kb. Longer times may also be helpful in later cycles when product concentration exceeds enzyme concentration and when dNTP and/or primer depletion may become limiting.
MAGNESIUM Buffers often contain Mg2+ (from MgCl2 or MgSO4) as a necessary cofactor for enzyme activity. Taq polymerase is particularly sensitive to Mg2+ concentration. At low Mg2+, Taq polymerase activity (correct complementary base pairing) is high, but polymerization rate is low. Conversely, at high Mg2+, the polymerization rate is high but the accuracy is low. Reduce concentration to prevent non-specific and undesirable PCR products. Increase to attain more product. EDTA chelates Mg2+ and can change the Mg2+ concentration.
PCR BUFFER Higher concentration of PCR buffer may be used to improve efficiency.
pH The 10X buffer contains a buffering agent (usually Tris-HCl) to maintain constant pH. The ideal pH for PCR is 8.4. For long templates, a higher pH (pH 9.0) is suggested. The pH of the Tris buffer in the reaction mix will decrease in high temperatures. The lower pH may cause depurination of the template, resulting in a lower yield of amplicons.
POLYMERASES Taq DNA polymerase has a higher error rate (no proofreading 3' to 5' exonuclease activity) than Pfx. Use Pfx if high fidelity is needed. Taq tends to add non-templated A at the 3' end. Extra enzyme may be added to improve efficiency (since Taq may be damaged in repeated cycling), but this may increase non-specific PCR products.
PRIMER CONCENTRATION Primer concentration in a common PCR reaction is about 100-500 nM per primer. Increasing primer concentration may improve the outcome of the PCR reaction and should be considered as a way to optimize PCR reactions. High primer concentrations may inhibit the reaction. Example: How do I make a 10uM stock solution starting with 24 nmol of oligo - Start by converting uM into nmol/ml: 10uM = 10 umol/L or 10 nmol/ml - Then figure out what volume you need to resuspend your oligo in based on your starting amount: 10 nmol/ml = 24 nmol/ X ml, solving for X = 2.4 ml or 2400 ul - To make a 10 uM stock solution, you would resuspend 24 nmol of oligo in 2400 ul. If you use 10 ul of a 10 uM stock solution in a 100-ul reaction, the final oligo concentration will be 1uM. Use the same amount for BOTH primers in your PCR. Up to 3 uM primers may be used. High primer to template ratio may result in non-specific amplification and primer-dimer formation. Store primers in small aliquots to avoid multiple freeze-thaw cycles.
PRIMER SEQUENCE Generally, primers used are 18-28 nt in length. This provides for practical annealing temperatures. Primers should avoid stretches of polybase sequences (e.g., poly dG) or repeating motifs -- these can hybridize inappropriately on the template. Aim for 50% GC content. High GC content results in the formation of stable imperfect hybrids while high AT content depresses the Tm of perfectly matched hybrids. If possible, the 3' end of the primer should be rich in GC bases (GC clamp) to enhance annealing of the end which will be extended. Inverted repeat sequences should be avoided to prevent formation of secondary structure in the primer which may prevent hybridization to template. Sequences complementary to other primers used in the PCR should be avoided so as to prevent hybridization between primers (primer dimers). Primer pairs should have compatible Tm (within 5 degrees). When adding sequences to the 5' end of the primer to create a restriction site, it is important to include a few extra bases (2-6 bases) to serve as a clamp to keep the 5' ends from breathing during digestion.
SALT Enzyme function and formation of primer-template hybrids require salt and buffer. Salt concentrations that maximize enzyme activity are generally sufficient to shield phosphate backbone repulsion. Buffers often contain 50mM KCl to provide the correct ionic strength.
TEMPLATE For most PCR reactions, template should be present at 10 to 1000 copies per reaction -- approximately 5-100 ng of tempate DNA. To reduce the likelihood of error by Taq DNA polymerase, a higher DNA concentration can be used. Too much template may increase the amount of contaminants and reduce efficiency.
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PCR and RTPCR Dyes

Dye Excitation Emission Application
Acridine 362 462 Primer Labeling
AMCA-S 353 437Primer Labeling
BODIPY® 493/503 500506Primer Labeling
BODIPY® 530/550 534554Primer Labeling
BODIPY® 558/569 559568Primer Labeling
BODIPY® 564/570 563 569 Primer Labeling
BODIPY® 581/591 581 591 Primer Labeling
BODIPY® 630/650-X 625 640 Primer Labeling
BODIPY® FL 502 513 Primer Labeling
BODIPY® FL-X 504 510 Primer Labeling
BODIPY® R6G 528 547 Primer Labeling
BODIPY® R6G-X 529 547 Primer Labeling
BODIPY® TMR 544 570 Primer Labeling
BODIPY® TR-X 589 617 Primer Labeling
Cascade Blue® 396 410 Primer Labeling
CC2-DMPE (no FRET) 400 460 Primer Labeling
CCF2 (FRET) 408 530 Primer Labeling
Coumarin (no FRET) 408 530 Primer Labeling
Cycle 3 GFP 395 507 395 445 Primer Labeling
D2-PA 750 507 Primer Labeling
D3-PA 685 706 Primer Labeling
D4-PA 650 670 Primer Labeling
DiSBAC (FRET) 400 580 Primer Labeling
FAM 492 520 Primer Labeling
Fluorescein 494 520 Primer Labeling
Fluorescein (FRET) 408 520 Primer Labeling
HEX 535 556 Primer Labeling
JOE 520 548 Primer Labeling
Marina Blue 362 459 Primer Labeling
Oregon Green® 488  495 521 Primer Labeling
Oregon Green® 488-X  494 517 Primer Labeling
Oregon Green® 500  408 520 Primer Labeling
Oregon Green® 514  535 556 Primer Labeling
Pacific Blue  520 548 Primer Labeling
Rhodamine  362 459 Primer Labeling
Rhodamine Green  495 521 Primer Labeling
Rhodamine Green-X  494 517 Primer Labeling
Rhodamine Red-X  499 519 Primer Labeling
Rhodol Green  506 526 Primer Labeling
Terbium Chelates  416 451 Primer Labeling
Vivid Blue  572 596 Primer Labeling
Vivid Cyan  504 532 Primer Labeling
Vivid Green  503 528 Primer Labeling
Vivid Red  560 580 Primer Labeling
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qPCR Troubleshooting

qPCR Problem Possible Cause Suggested Solution
No amplification curves seen after data analysis. There is no PCR product. Verify whether your PCR worked by running a gel.
No product is seen on a gel.Incorrect cycling parameters, annealing temperature or other parameters may have been used. Check your cycling parameters as you would a standard PCR.
A band is seen on the gel but the analysis does not show any curves. Wrong settings on the machine. Check the acquisition points in your program. Data is usually collected at the annealing step for probe-based assays, and at the extension step for other chemistries (SYBR®). If the collection points are set properly and a band appears on the gel, check the reference dye.
A band is seen on the gel but the analysis does not show any curves. The wrong channel filter was chosen for the fluorophore being used. Check the filter/channel designations for your machine. The emission wavelength for your fluorophore can be detected in a specific channel.
A band is seen on the gel but the analysis does not show any curvesThe machine may be incapable of measuring that dye as well as the reference dye. Check to see if machine is compatible with the dye and fluorophores being used.
A band is seen on the gel but the analysis does not show any curves. The light source (Halogen lamp or laser) is not working properly. Look at the other options first, especially if you have recently calibrated the machine or replaced the lamp. Recalibrate the machine or use the reference plates according to manufacturer’s recommendations.
No melting curve or the lines are flat all the way across. There is no PCR product. f the lines are flat, or there are no lines at all, check for presence of any PCR product by running a gel. The gel may indicate whether product was made or whether only a primer dimer was made. In the case of SYBR® green, absence of a melting curve points to PCR failure because even primer dimers give a melting profile.
Multiple peaks/bumps are present in the plot. Primer dimers are present. Improve the stringency by raising the annealing temperature or lowering magnesium concentration. If the annealing temperature is increased above 60°C, a two-step protocol can be used in place of a three-step protocol. Try a hot-start reaction. We recommend the Platinum® DNA Polymerases.
Multiple peaks/bumps are present in the plot. Primers are amplifying multiple genuine products (e.g., variants in cDNA template). With variants from a cDNA, you may have to redesign the primers. If you run the PCR product on a gel, a significant difference in size (equal to an extra intron) will show up. The Tm of those products may be above 80°C.
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Troubleshooting

RT-PCR Problem Possible Cause Suggested Solution
Little or no RT-PCR product visible after agarose gel analysis. 
RNA was degraded. 
Analyze RNA on a denaturing gel before use to verify integrity. Use aseptic technique for RNA isolation. Process tissue immediately after removal from animal. Store RNA in 100% formamide. If using placental RNase inhibitor, do not heat >45°C or use >pH 8.0 or inhibitor may release any bound RNases. Also, DTT must be present when the RNase inhibitor is added at =0.8 mM DTT. 
Little or no RT-PCR product visible after agarose gel analysis.
RNA contained an inhibitor of RT.
Remove inhibitor by ethanol precipitation of the RNA. Include a 70% (v/v) ethanol wash of the RNA pellet. Glycogen (0.25 µg to 0.4 µg/µl) can be included to aid in RNA recovery for small samples. Inhibitors of RT include: SDS, EDTA, glycerol, sodium pyrophosphate, spermidine, formamide, and guanidinium salts. Test for inhibitors by mixing a control RNA with the sample and comparing yields to control RNA reaction. 
Little or no RT-PCR product visible after agarose gel analysis. 
Polysaccharide coprecipitation of RNA. 
Precipitate RNA with lithium chloride to remove polysaccharides.
Little or no RT-PCR product visible after agarose gel analysis. Primer used for first-strand cDNA synthesis did not anneal well. Be sure annealing temperature is appropriate for your primer. For random hexamers, a 10 min. incubation at 25°C is recommended before incubating at reaction temperature. For gene-specific primers (GSP), try another GSP or switch to oligo(dT) or random hexamers. Make sure GSP is the antisense sequence. 
Little or no RT-PCR product visible after agarose gel analysis.Not enough starting RNA. Increase the amount of RNA. For <50 ng RNA, use 0.1 µg to 0.5 µg acetylated BSA or 40 units of RNaseOUT™ Ribonuclease Inhibitor in first-strand cDNA synthesis (3,4). To maximize RT-PCR sensitivity, use the SuperScript™ III First-Strand Synthesis System for RT-PCR.   
Little or no RT-PCR product visible after agarose gel analysis. RNA template had high secondary structure. Denature/anneal RNA and primers in the absence of salts and buffer. Raise the RT reaction temperature up to 55°C for SuperScript™ III or up to 65°C for ThermoScript™ RT (5). Note: Do not use oligo(dT) as a primer over 60°C and choose a GSP that will anneal at your reaction temperature. For RT-PCR products >1 kb, keep reaction temperature = 65°C. Do not use M-MLV RT above 37°C. Use random hexamers in the first-strand reaction if full-length cDNA is not needed. 
Little or no RT-PCR product visible after agarose gel analysis The primers or template are sensitive to remaining RNA template. Treat first-strand cDNA with RNase H before PCR.
Little or no RT-PCR product visible after agarose gel analysis.  
Target not expressed in tissue analyzed. 
Try a different target or tissue. 
Little or no RT-PCR product visible after agarose gel analysis. 
PCR did not work. For two-step RT-PCR, do not use more than 1/5 of the RT reaction in the PCR step.
Little or no PCR product visible after agarose gel analysis
Poor PCR primer design.
Avoid complementary sequences at the 3´ end of primers. Avoid sequences that can form internal hairpin structures. Design primers with similar Tms.
Little or no PCR product visible after agarose gel analysis
DNA contains inhibitors.
Reagents such as DMSO, SDS, and formamide can inhibit Taq DNA polymerase. If inhibitor contamination is suspected, ethanol precipitate the DNA sample.
Little or no PCR product visible after agarose gel analysis
GC-rich template
For templates >50% GC content, use PCRx Enhancer Solution, or the Accuprime GC-Rich DNA polymerase.
Little or no PCR product visible after agarose gel analysis
Template concentration is too low.
Start with 1000 copies of the target sequence to obtain a signal in 25 to 30 cycles.
Little or no PCR product visible after agarose gel analysis
Magnesium concentration is too low.
Determine the optimal magnesium concentration for each template and primer pair by performing a reaction series from 1 mM to 3 mM in 0.5 mM increments. Note: Use 3 mM to 5 mM magnesium for real-time quantitative PCR.
Little or no PCR product visible after agarose gel analysis
Annealing temperature is too high.
Estimate the Tm using a computer program or equation and set the annealing temperature 5°C below the Tm. Since these equations estimate Tm values, the true annealing temperature may actually be higher or lower.
Little or no PCR product visible after agarose gel analysis
Primer concentration is too low.
Optimal primer concentration is between 0.1 µM to 0.5 µM. To accurately determine primer concentration, read the optical density at 260 nm (OD260). Then, calculate the concentration using the absorbance and the extinction coefficient.
Unexpected bands after gel analysis.
Non-specific annealing of primers to templates.
Use a GSP instead of random hexamers or oligo(dT) for first-strand synthesis. Try a GSP that allows cDNA synthesis at high temperatures
Unexpected bands after gel analysis.
Poor GSP design.
Follow the same rules as described for amplification primers.
Unexpected bands after gel analysis.
Genomic DNA contamination of RNA.
Treat RNA with DNase I, Amplification Grade. Check for DNA contamination with a control reaction without RT.
Unexpected bands after gel analysis.
Primer-dimer formation. 
Design primers without complementary sequences at the 3´ ends.
Unexpected bands after gel analysis. 
Non-specific annealing of primers to template.
Increase the annealing temperature in 2°C to 5°C increments and minimize the annealing time. Use higher annealing temperatures for the first few cycles, followed by lower annealing temperatures. Use Platinum® Ta q DNA Polymerase for automatic hot-start PCR (9). Avoid 2 or 3 dGs or dCs at the 3´ end of primers. 
Unexpected bands after gel analysis.
Magnesium concentration is too high.
Optimize magnesium concentration for each template and primer combination.
 Unexpected bands after gel analysis. 
Primer mispriming due to amplification from complex templates.
Use nested primers or touchdown PCR.
Unexpected bands after gel analysis.
Contaminating DNA from an exogenous source.
Use aerosol-resistant tips and UDG.
Unexpected bands after gel analysis.
Primer binding sites are inaccessible due to secondary structure. 
For templates >50% GC content use (1X-3X) PCRx Enhancer Solution, Accuprime GC-Rich DNA Polymerase, or additives.
PCR induced errors found in product sequence      
Polymerase has low fidelity.Use a proofreading thermostable polymerase such as Platinum® Pfx DNA Polymerase. Note: The conditions for using Platinum® Pfx DNA Polymerase differ from other proofreading enzymes. Use a lower magnesium concentration (1 mM final), lower annealing temperature, and fewer units of enzyme (1.25 units). In addition, if products amplified with Platinum® Pfx DNA Polymerase remain in the well during electrophoresis, add SDS to the loading buffer to 0.1%. 
PCR induced errors found in product sequence
Too many cycles.
Reduce cycle number.
PCR induced errors found in product sequence 
The concentration of all four deoxynucleotides is not equal. 
Prepare a new deoxynucleotide mix and ensure that the concentration of all four nucleotides is equal, or use a prepared mix.
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References

  1.    Fox, D.K. (1998) Focus® 20: 84.

  2.    Sewall, A. (1999) Focus® 21: 2.

  3.    Breslauer, K.J. et al. (1986) Proc. Natl. Acad. Sci. USA. 83: 3746-3750.

  4.    Rychlik, W. et al. (1990) Nucleic Acids Res. 18: 6409-6412.
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