When you just graduated and entered the job, you are faced with a bunch of nouns facing real-time quantitative PCR experiments. Are you confused about where to start? What is the meaning of amplification curve, standard curve, threshold, CT value, melting curve and baseline? The fluorescent images from those experiments are too bright, but I can't seem to identify them. Today, we will take you to learn these knowledge and concepts in two parts: technical terms and frequently asked questions.
Part I Professional Terms
1. Amplification curve
The amplification curve refers to the curve where the cycle number is the abscissa and the real-time fluorescence intensity during the reaction is the ordinate during the PCR.
2. Baseline
Baseline refers to little change in fluorescent signal during the first few cycles of a PCR amplification reaction. The signal level shows close to a straight line, which is the baseline.
3. Fluorescence threshold setting
Typically, the fluorescence signal of the first 15 cycles of the PCR reaction is used as the fluorescence background signal, and the fluorescence threshold is 10 times the standard deviation of the fluorescence signal during 3-15 cycles of PCR, and the fluorescence threshold is set during the exponential phase of PCR amplification. Typically, each instrument has its fluorescence threshold before use.
4. CT value
The CT value represents the number of cycles that will occur for each PCR reaction tube when the fluorescent signal reaches the set threshold. From research, we know that there is a linear relationship between the CT value of each template and the logarithm of the starting copy number of that template. The higher the initial copy number, the smaller the CT value, and vice versa. Using standards with known starting copy numbers, a calibration curve can be made where the abscissa represents the logarithm of the starting copy number, while the ordinate represents the CT value. Therefore, as long as the CT value of the unknown sample is obtained, the initial copy number of the sample can be calculated from the standard curve.
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There are several indicators to judge whether the amplification curve is good or not:
Answer: The inflection point of the curve is obvious, especially the exponential phase of the low-heavy fraction sample is obvious, the overall parallelism of the amplification curve is very good, the baseline is flat, there is no rising phenomenon, and the amplification curve of the low-level exponential phase concentration sample is very good. obvious.
B: The slope of the exponential phase of the curve is directly proportional to the amplification efficiency, the larger the slope, the higher the amplification efficiency.
C: The standard baseline is flat or slightly decreasing, with no apparent upward trend.
D: The parallelism of the amplification curves for each tube is good, indicating that the amplification efficiency of each reaction tube is similar.
5. Melting curve
When the PCR product is heated, as the temperature rises, the double-stranded amplification product gradually dissociates, resulting in a decrease in fluorescence intensity. When a certain temperature is reached, a large amount of product separates, resulting in a sharp decrease in fluorescence. Using this function together with different TM values for different PCR products also differs in the temperature at which the fluorescent signal rapidly decreases, which may be a good way to identify PCR specificity.
6. Melting curve (using logarithmic curve)
The peak graph is formed by the logarithm of the melting curve to more intuitively display the situation of product fragments. Since the melting temperature is the TM value of a DNA fragment, certain parameters affecting the TM value of a DNA fragment can be determined, such as fragment size, GC content, etc. Generally, according to our primer design principle, it is usually said that the length of the amplified product is in the range of 80-300bp, and the melting temperature should be between 80°C and 90°C.
A: If there is only one main peak between 80°C and 90°C, it means that the real-time PCR is perfect.
B: If the main peak appears between 80°C and 90°C, and the impurity peak appears below 80°C, the primer-dimer is basically considered. This is a good option to try to solve by raising the annealing temperature.
C: If the main peak appears between 80°C and 90°C, and a wandering peak appears when the temperature increases, DNA contamination is basically considered. And the DNA needs to be removed in the initial stages of the experiment.
7. Standard curve line
Standard standards were diluted to different concentrations and used as templates for PCR reactions. The standard curve is plotted with the logarithm of the standard copy number as the abscissa and the measured CT value as the ordinate. When quantifying an unknown sample, the copy number of the sample can be obtained from the standard curve based on the CT value of the unknown sample. Standard curves are very important for absolute quantification.
the second part. common problem
Q1: What is the difference between RT-PCR, QPCR, real-time PCR and real-time RT-PCR?
A1: RT-PCR is reverse transcription PCR, which is a widely used variant of polymerase chain reaction. In RT-PCR, the RNA strand is reverse transcribed into complementary DNA, which is then used as a template for PCR to amplify the DNA by PCR.
Real-time PCR and QPCR are the same thing, both are real-time quantitative PCR, which means that there is real-time recording of data at each cycle during the PCR process. In this way, the number of startup templates can be precisely analyzed.
Although both real-time PCR and reverse-transcription PCR appear to be abbreviated as RT-PCR, the international convention is that RT-PCR refers specifically to reverse-transcription PCR, while real-time PCR is often abbreviated as qPCR (Quantitative Real-True Real-True Real PCR ) - time PCR).
Real-time RT-PCR (RT-QPCR) is a type of reverse transcription PCR that combines fluorescent quantitative techniques: first reverse transcription from RNA to obtain cDNA (RT), followed by quantitative analysis using real-time PCR (QPCR).
Q2: Why is the length of the amplified product fragment of fluorescent quantitative PCR controlled within the range of 80-300bp?
A2: The length of each gene sequence is different, some of them are several Kb and hundreds of BP. However, when we design the primer, we only need to require the length of the product to be 80-300bp, and too short or too long is not suitable for quantitative PCR detection.
Product fragments are too short to be distinguished from primer-dimers. The length of the primer-dimer is about 30-40bp, when it is less than 80bp, it is difficult to distinguish whether it is a primer-dimer or a product. If the product fragment is too long, exceeding 300bp, it will easily lead to low amplification efficiency, and the amount of gene cannot be effectively detected.
For example, when you count the number of people in a classroom, you only need to count how many mouths there are. The same is true when testing genes. You only need to detect a certain sequence of a gene to represent the entire sequence. It's easy to make a mistake if you're counting both mouths and noses, ears, and glasses to count people.
Q3: What is the optimal length for the primer design?
A3: Generally speaking, primer lengths in the range of 20-24bp are good. Of course, we must pay attention to the TM value of the primer when designing the primer, because it is related to the optimal annealing temperature. After a lot of experiments, it has been proved that 60°C is a good TM value. Low annealing temperature can easily lead to non-specific amplification, while high annealing temperature usually leads to low amplification efficiency, late peak of amplification curve and delayed CT value.
Question 4: Does the amount of sample collected affect the experimental results?
A4: No. Obviously, the more samples collected, the more RNA extracted, the more cDNA, and the more target fragments there will be. For absolute quantification, it is necessary to calculate the copy number of the target fragment, and the amount of sample collected will definitely affect the experimental results. For example, the detection of hepatitis C virus HBV in the blood is to detect the HBV content in a certain amount (1ml) of blood.
For relative quantification commonly used in scientific research, the number of samples has nothing to do with the experimental results, because relative quantification refers to the comparison between the target gene and the reference gene. Just please consider them as upstream and downstream fragments present in the same nucleic acid strand. If the sample size is large, both reference and target genes are increased in equal proportions at the same time, which does not affect the results.
Q5: Will the efficiency of reverse transcriptase affect the experimental results?
A5: Same as above. Note that we want higher RT efficiency, but we prefer that the RT enzyme be relatively stable and obtain uniform results. This will be a test of the optimized capabilities of the major companies' reverse transcription suites.
Question 6: Does the efficiency of TAQ enzyme affect the experimental results?
A6: The efficiency of TAQ enzyme is relatively large. Typically, hot-start taq enzymes are required and are relatively efficient. For commercial fluorescence quantitative kits, each manufacturer will optimize the efficiency of the best state according to their own products close to 100%, if the efficiency is too low, the experimental results cannot be obtained. For different companies' products, this is a measure of quality.
Question 7: Does the amount of fluorescent dye affect the experimental results?
A7: Yes, it will. If the fluorochrome is too saturated, it can cause noise interference in some instruments. If the fluorochrome is not saturated, and the fluorescence value is too low, it will enter a plateau phase early and the amplification curve will be flat. In the fluorescence quantitative experiment, the CT value is mainly seen, so it is not important for the late amplification curve to enter the plateau stage, but the picture is not beautiful enough. If you had to choose, start by choosing a fluorochrome that is slightly less saturated. However, when using the same product from the same company, its effect is basically negligible.
Question 8: Will the transmission of the PCR tube affect the experimental results?
A8: Yes. However, for the same batch of consumables from the same manufacturer, this effect can be ignored. This is a good choice and it is best to use a 96-well plate with a high-permeability membrane to minimize the effect of the light transmittance of the consumables.
Q9: Will errors during operation affect the experimental results?
A9: The influence of the operation process is mainly reflected in the uniformity. Homogeneity means that all components in the system are evenly mixed together, and flash centrifugation can solve this problem. In addition, for beginners, it is best to adjust the PCR system to be more than 20 inches, and a system that is too small is more prone to errors. If the annealing temperature is optimized correctly, the effect of primer concentration on CT is minimized. And you'll know that some operational errors (such as primer concentration) can be avoided by optimizing the annealing temperature.
Summarize
The above are some questions and questions that novices often encounter during the experiment, we hope these questions can help you solve some confusion. Experimentation is the process of generating chaos and solving problems, hopefully you get something out of it. Thanks for reading and we'll see you next time!
