The threshold level and C q value on a real-time PCR amplification curve. C q values are inverse to the amount of target nucleic acid that is in your sample, and correlate to the number of target copies in your sample. Lower C q values typically below 29 cycles indicate high amounts of the target sequence. Higher C q values above 38 cycles mean lower amounts of your target nucleic acid.
High C q values can also indicate problems with the target or the PCR set-up, as outlined later in the pitfalls section of this article. Your PCR instrument will collect fluorescence data during each cycle. The threshold level will be just above this, but at the point where your samples start moving into the exponential phase of PCR amplification. Today, computer software calculates this exact point and all modern real-time cyclers have an automatic threshold line setting.
In this way, C q values are usually consistent across replicates in real-time PCR. By the time the PCR reaction endpoint is reached, accumulated inhibitors, inactivated polymerases and limiting reagents create a lot of variation in endpoint values, and this is why conventional PCR cannot be used quantitatively. Many factors can affect your C q values. Some differences in C q values between your samples will be due to biological events e. The most common pitfall areas are:.
Fluorescence emission can be affected by pH and salt concentration in a solution. Any change in fluorescence emission will naturally change your C q values. Therefore, make sure you only use high-quality PCR components and if using homemade solutions, check the pH, and monitor salt precipitation before each experiment. PCR reaction efficiency is dependent on the master mix performance, the specificity of the primers, the primer annealing temperature, and the sample quality.
Perfect PCR efficiency coincides with a change of 3. To determine the PCR efficiency for each primer pair, run serial dilutions of your template with five fold dilution steps, and calculate the R 2 , a statistical measure that describes how well one value can predict another.
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Muromachi Kikai Co. Geneer Inc. AH diagnostics Oy Viikinkaari 4. AH diagnostics AB Vallgatan 9. AH Diagnostics Fjellgata 1. This is because it takes fewer PCR cycles for that product to be detected over the background signal. You will also often hear the term cycle quantification Cq , maybe even crossing point Cp or take-off point TOP. Basically, these terms all refer to the same value as the Ct value.
Discrepancies in nomenclature have been historically inconsistent, with different qPCR manufacturers using different variations. Therefore, most people actually prefer the term Cq, as opposed to Ct.
Hi , thanks for these nice information , but can you put references in your writings? Your information is simple and valuable. Wonderfully concise explanation my dude. With cT and cQ being essentially the same thing, and them both referring to a cycle when fluorescence has increased significantly, could you explain what a cQ value would mean as a non integer?
A CQ value of 9. The values are never integers, but the same concept applies. Since the cQ value is on the log scale, any subtle differences in value could translate to large differences in gene expression values. I hope that makes sense? Best wishes, Steven. Hi Ruth, Many thanks for your message. I am glad you found it useful. Hi Steven, nice article! However, I find the second figure not quite correct.
The threshold value should be set in the exponential phase as written quite correctly. In the figure however, this is clearly not done correctly, since the threshold line is set at the beginning of the exponential phase.
Hi Lorenz, Many thanks for your comment and the point about the figure. As you can see, I am not the best at drawing figures haha, I will amend this shortly :.
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