SYBR Green qPCR protocol
Steps:
Check literature and databases (such as http://www.rtprimerdb.org) for preexisting primers for the gene under study.
Choose a target sequence.
Design primer and check for specificity.
Validate the primers and optimize the protocol
Check for preexisting primers
Check the literature and databases for preexisting primers which greatly saves time.
Choose a target sequence
Target sequence must ideally be 75–200 bp. Short PCR products are amplified with higher efficiency, but it should be at least 75 bp long to easily distinguish it from any primer-dimers that is formed.
Avoid regions that form secondary structure at the annealing temperature, when possible. Use programs such as mfold to predict secondary structure.
Avoid regions with long (>=4) repeats of single bases.
Choose a region that has a GC content of 50–60%
Design primer and check for specificity
Design primers that have a GC content of 50–60%.
Tm should be between 50 and 65°C. Use 50 mM for salt concentration and 300 nM for oligonucleotide concentration during online calculation of Tm.
Avoid secondary structure; adjust primer locations so they are located outside secondary structure in the target sequence, if required
Avoid repeats of Gs or Cs longer than 3 bases. Place Gs and Cs on the ends of the primers
Check the sequences for primer-dimer formation of forward and reverse primers by ensuring no 3' complementarity.
Verify specificity of primer by using BLAST.
Validating the primers and optimize the protocol
To achieve accurate template quantification in a qPCR assay, each reaction must efficiently amplify a single product, and amplification efficiency must be independent of template concentration and the amplification of other templates.
Determining Reaction Efficiency: Using a Standard Curve
A common method for validating qPCR assays involves the construction of a standard curve, enabling the determination of the efficiency of a qPCR assay. The percentage efficiency of the assay should be 90–105%, the R2 of the standard curve should be >0.980 (or r > –10.990), and the quantification cycle (Cq) values of the replicates should all be similar.
The standard curve is constructed by plotting the log of the starting quantity of template (or the dilution factor, for unknown quantities) against the CT value obtained during amplification of each dilution. The equation of the linear regression line, along with Pearson’s correlation coefficient (r) or the coefficient of determination (R2), can then be used to evaluate whether your qPCR assay is optimized. The dilution series will produce amplification curves that are evenly spaced (Figure 1A)
Figure. 1 Generating a standard curve to assess reaction optimization. A standard curve was generated using a 10-fold dilution of a template. Each dilution was assayed in triplicate. (A) Amplification curves of the dilution series. (B) Standard curve with the CT plotted against the log of the starting quantity of template for each dilution.
If perfect doubling occurs with each amplification cycle, the spacing of the fluorescence curves will be determined by the equation 2n = dilution factor, where n is the number of cycles between curves at the fluorescence threshold. For instance, with a 10-fold serial dilution of DNA, 2n = 10. Therefore, n = 3.32, and the CT values should be separated by 3.32 cycles. Evenly spaced amplification curves will produce a linear standard curve, as shown in Figure 1B. The equation and R2 value of the linear regression line are important.
Note: The absolute value of the slope is the same as the even space between the amplification curves (which is 3.32).
Example
An experiment can be constructed by using various dilutions of template cDNA (10 to 105) and running qPCR for the gene of interest with no template controls in triplicate. The resulting CT values were plotted against the various respective dilutions to obtain Figure 2.
Figure 2. Standard curve for a gene used for example, with the CT plotted against the log of the starting quantity of template for each dilution.
The R2 as seen in Figure 2 should be > 0.98 and the efficiency (E) is calculated using the formula:
Where slope is the coefficient of x which is -3.1716 as in Figure 2.
Therefore E = 10 – (1/-c) = 2.07 (approximately = 2)
which confirms 2-3.1716 =10.04(dilution factor)
And %E = (E-1) X 100 = 106.68%
An efficiency close to 100% (90–105%) is the best indicator of a robust, reproducible assay. Low reaction efficiencies may be caused by poor primer design or by suboptimal reaction conditions. Reaction efficiencies >100% may indicate pipetting error in your serial dilutions or co-amplification of nonspecific products, such as primer dimers.
This experiment will also help us determine the template dilution suitable for running our qPCR experiments.
qPCR Reaction mix recipe (10µl)
10mM forward primer = 0.4 µl
10mM reverse primer = 0.4 µl
cDNA = 1 µl (Add finally to the wells and not to the mastermix)
2 X iTaq Universal SYBR = 5 µl
Green Supermix
Double distilled H2O = 3.2 µl
Note: Always mix 2 X iTaq Universal SYBR and ddH2O then split and add the respective primers for control and study genes. Also prepare a little extra to account for pipetting error during loading of samples. Add the template DNA finally to the wells and then centrifuge.
Optional optimization
qPCR Assay Specificity Verification by Melt Curve
Melt curve analysis can be used to identify different PCR products, including nonspecific products and primer-dimers when the fluorescence of the reporter chemistry depends on the presence of dsDNA, as with SYBR® Green dye. This property is valuable because the presence of secondary nonspecific products and primer-dimers can severely reduce the amplification efficiency and, ultimately, the accuracy of the qPCR assay. Primer-dimers can also limit the dynamic range of the desired standard curve due to competition for reaction components during amplification.
Figure 3. Melt-curve analysis of reaction product from a SYBR Green assay. The melt-curve analysis function of real-time instruments can be used to distinguish specific products from nonspecific products. (A) The negative first derivative of the change in fluorescence is plotted as a function temperature. The two peaks indicate the Tm values of two PCR products. (B) Gel analysis of the qPCR products. Lane 1, AmpliSize® 50–2,000 base pairs (bp) molecular ruler; lanes 2 and 3, two replicates of qPCR product from the reaction shown in (A). The two PCR products are revealed by separate bands in the gel.
The melt peak distinguishes specific products from other products that melt at different temperatures, such as primer-dimers. Because of their small size, primer-dimers usually melt at lower temperatures than the desired product, whereas nonspecific amplification can result in PCR products that melt at temperatures above or below that of the desired product. In the melt curve in Figure 3, the peak at 89°C represents the target qPCR assay product and corresponds to the upper band in lanes 2 and 3 on the gel. The peak at 78°C represents the nonspecific product and corresponds to the lower band in lanes 2 and 3 on the gel.
Annealing Temperature Optimization
The annealing temperature of a qPCR assay is one of the most critical parameters for reaction specificity. Setting the annealing temperature too low may lead to amplification of nonspecific PCR products. Conversely, setting the annealing temperature too high may reduce the yield of a desired PCR product. Even after calculating the Tm of a primer, you may need to determine the annealing temperature empirically by repeating a reaction at many different temperatures. Similar time-consuming tests may also be required to optimize the denaturation temperature.
To find the optimal annealing temperature for your qPCR assay, test a range of temperatures above and below the calculated Tm of the primers. The optimal annealing temperature is the one that results in the lowest Cq with no nonspecific amplification. The results of a sample annealing temperature optimization experiment are shown in Figure 4.
Figure 4. Annealing temperature optimization. An annealing temperature gradient of 55°C to 72°C was performed on the Chromo4 system. The 62.2°C reaction gave the lowest CT value and was selected as the optimal annealing temperature for this assay.