All rights reserved. For specific trademark and licensing information, see www. Order by stock part number ». Get Help EN. Submit question. Apply Close. Chat now. Toggle navigation. Steps for a successful qPCR experiment. Print Page. Assay design A well-designed assay begins with an understanding of the gene of interest, including knowledge of the transcript variants and their exon organization.
The gradient feature allows you to test a range of temperatures simultaneously, so the annealing temperature can be optimized in a single experiment. To find the optimal annealing temperature for your qPCR assay, test a range of temperatures above and below the calculated T m 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 2.
Annealing temperature optimization. The To do this, use the melt curve function on your real-time instrument and also run products on an agarose gel.
Figure 3 shows the melt curve and agarose gel analyses from the annealing temperature optimization experiment shown in Figure 2. The reaction with annealing temperature at A , melt curve analysis with a plot of the first derivative of the change in fluorescence intensity as a function of temperature. B , agarose gel analysis of the reaction products. Lane 1, molecular weight markers, — 1, bp in bp increments, 1, bp, and 1, bp.
Lane 2, single PCR product corresponding to the peak observed in A. Multiplex qPCR Assay Validation To obtain accurate results from a multiplex assay, you must ensure that no target's amplification influences that of another. To determine whether all reactions proceed independently in the multiplex assay, run singleplex and multiplex assays for your targets in the same plate and compare the Cq values. The values obtained for a given target in the singleplex and multiplex assays should not differ significantly.
If the Cq values from the singleplex and multiplex reactions are significantly different, you will need to optimize your reactions. Multiplex qPCR Assay Optimization If a multiplex reaction is not optimized, the amplification of a more efficient or more abundant target can inhibit that of a less efficient or less abundant target.
This occurs because reaction components such as DNA polymerase, dNTPs, and MgCl2 become limiting in later cycles, and the amplification of the less efficient or less abundant target is compromised. Inhibition of a target's amplification will cause its Cq to be delayed in a multiplex qPCR assay relative to that in the singleplex assay. Gudnason H et al. Nucleic Acids Res 35, e PMID: Juskowiak B Nucleic acid-based fluorescent probes and their analytical potential.
Anal Bioanal Chem , — However, the quantities used are always identical for the same sample, making it possible to compare Cq values in order to assess the effect of amplicon size. Plasmids were linearized with Bam HI enzyme Roche Diagnostics, Mannheim, Germany and products of linearization were purified with the last steps of a Phenol-Chloroform extraction Sambrook et al. Probes were synthesized by Eurogentec Seraing, Belgium. Primers were chosen with Primer Express program v2. Primer and probe sequences are presented in table 1.
At The wells were covered by optical caps Eurogentec. The plates were centrifuged rpm, 10 s to eliminate any air bubbles at the bottom of the wells. At the end of the run, the baseline the normalized fluorescent signal before exponential PCR amplification occurs was adjusted and a threshold level of fluorescence was fixed in the middle of the linear region of the amplification curve represented in a semi-logarithmic graph Y-axis with log fluorescence level as a function of cycles.
This allowed the Cq values Quantitation Cycle, sometimes also referred to as the Ct value, for Cycle threshold of the different samples to be compared. The primers were developed in order to progressively increase the amplicon size. The forward primer was kept close to the probe to allow the probe to be rapidly degraded by the exonuclease activity of the Taq polymerase during the elongation step.
Here too, the amplicons were progressively increased to reach the following sizes: 81, , , , , , and bp Figure 3b. This treatment led to a cleavage of DNA strands and a distribution in favor of small fragments;. Kernels of an organic soybean were also used. This was considered to be like a pure nucleic acid material for which amplifications were deemed not to be subject to a matrix effect. Two different plasmids were used. The first plasmid, pl-Lec, contains a bp fragment of lectin gene and the second plasmid, pl-RR, contains a bp fragment of Roundup Ready construction including left border plant-P35S junction Windels et al.
Sequences of the largest fragments amplified with primers from this study were inside the cloned fragments. In order to compare the results, all amplifications for lectin or RR targets were performed simultaneously for a determined matrix. Comparison between the two chemistries was performed on identical quantities of DNA per sample and with the data from a same run. This was done by selecting the appropriate filters on real-time PCR apparatus and applying a dissociation protocol at the end of the run.
Cq values were determined by fixing a threshold level within the logarithmic part of the amplification curves. Means of Cq values are presented in the different tables. Earlier detection lower Cq value and a higher fluorescence level plateau phase were generally observed for shorter amplicons Figure 4. Another observation was the loss of efficiency when larger amplicons were targeted. The efficiency of amplification was evaluated through the raw data of fluorescence obtained for the construction of the amplification curves data accessible on the ABI real-time PCR device and calculated for three different samples: DNA from reference flour at 0.
For one of the targets RR target however, the bp fragment was sometimes detected earlier than the bp target. This was probably due to a better combination of primers for this bp target. Primers were selected according to Primer Express software requirements, so it was not always possible to change the position of reverse primers by exact steps of 40 bp. For quantitative PCR, standard amplicons range from 75— bp. It is unlikely that an amplicon will be too short.
The polymerization rate of the enzyme used in the reaction needs to be sufficient to copy the amplicon in its entirety, so for long amplicons, you may need to increase the extension cycle time. You will also need to optimize reaction component concentrations based on the amplicon size.
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