The fluorescence quantitative PCR instrument is a high-precision experimental instrument, the following is its general use:

Pre-experiment preparation
Sample collection and processing
Collect suitable samples according to the purpose of the test, such as blood, throat swabs, tissues, etc. in clinical diagnosis; collect cells, tissues or other biological samples according to the experimental design in scientific research experiments.
The samples are processed to extract nucleic acids (DNA or RNA). Taking RNA extraction as an example, the cells are usually lysed with reagents containing lysate to release RNA, and then impurities are removed by centrifugation, filtration and other steps, and then RNA is precipitated with isopropyl alcohol or ethanol, and finally RNA is dissolved with an appropriate amount of nuclease-free water, resulting in pure nucleic acid samples for the subsequent experiments.
Reagent preparation
Select suitable reagents for quantitative PCR, including reaction buffer, dNTPs (deoxyribonucleotide triphosphates), primers, probes (if TaqMan probe method is used) and heat-stable DNA polymerase (e.g. Taq enzyme).
Prepare the reaction system accurately according to the experimental design and sample size. Typically, the volume of the reaction system is between 10 – 50 μL. Mix the reagents proportionally according to the reagent instruction manual, e.g., this may include 1 – 2 μL of primer, 1 – 2 μL of probe, an appropriate amount of template DNA or RNA, a certain amount of enzyme and buffer, etc., and then top up to the required volume with nuclease-free water.
Instrument Warm-up and Inspection
Switch on the PCR instrument and warm up the instrument to the set reaction temperature, usually between 95 – 100°C. This process usually takes 10 – 30 minutes, depending on the instrument model.
Check that the optical system, heating and cooling modules are working properly. This can be confirmed by using the instrument’s own test programme or by checking the instrument status indicators. Also check that the reaction holes are clean and, if necessary, clean them with a special cleaning tool.

Sample Spiking
Labelling of reaction tubes
Use a labelling pen or label printer to clearly label the PCR reaction tubes (usually 0.2mL or 0.1mL thin-walled tubes) with the sample number, experimental group, etc. Make sure that the labels are clear and accurate to avoid confusion.
Sample addition
Carefully add the prepared reaction system into the labelled PCR reaction tubes, and then add the appropriate amount of nucleic acid template. Use a micropipette to add the sample, paying attention to accurate operation and avoiding air bubbles. After adding the sample, gently flick the reaction tube to mix the liquid thoroughly, but avoid spilling the liquid.
Instrument Settings
Programme Setting
According to the experimental requirements and the fluorescence quantitative PCR reagents used, set up the reaction programme. Generally, it includes three main stages: denaturation, annealing and extension.
Denaturation phase: the temperature is usually set at 94 – 98°C for 10 – 30 seconds. The purpose of this phase is to unravel the double-stranded DNA template or RNA-cDNA complex into a single strand in preparation for the subsequent primer binding.
Annealing phase: the temperature is set according to the Tm value of the primer (unwinding temperature), usually between 50 – 70°C for 10 – 30 seconds, to allow the primer to bind specifically to the single-stranded template.
Extension phase: The temperature is usually around 72°C and the time depends on the length of the amplified product, usually 30 – 60 seconds per 1000bp (base pairs), this phase is where the DNA polymerase synthesises a new DNA strand from dNTPs under the guidance of the primer.
The number of cycles is usually set between 30 – 45, depending on the experimental requirements and template concentration. At the same time, it is necessary to set the stage of fluorescence signal acquisition, which is usually carried out at the end of the extension stage to detect the amount of amplification products.
Fluorescence channel setting
According to the fluorescence wavelength of the fluorescent dye or probe used, set the corresponding fluorescence channel on the instrument. For example, if a SYBR Green fluorescent dye is used, set its corresponding fluorescence channel to detect the fluorescence signal after double-stranded DNA binding; in the case of a TaqMan probe, set the channel corresponding to the wavelength at which the fluorescent moiety of the probe emits.
Running the reaction
Starting the reaction
After confirming that the instrument settings are correct, place the reaction module with reaction tubes into the PCR instrument, close the lid, and start the reaction.
Monitoring the reaction process
During the reaction, the instrument will automatically control the temperature change and fluorescence signal acquisition according to the set programme. The process of the reaction can be viewed in real time through the instrument’s display or the connected computer software, including the temperature change curve and the fluorescence signal intensity change curve.
Analysis of Experimental Results
Data analysis method
After the reaction, the instrument will generate fluorescence signal data. This data is usually processed using quantitative PCR analysis software, and the most common analysis method is based on the Ct value (cycling threshold), which refers to the number of cycles that the fluorescence signal passes through when it reaches the set threshold value during PCR amplification.
Based on the Ct value, the initial amount of the target nucleic acid in the sample can be calculated, usually by relative or absolute quantification. Relative quantification is to determine the relative expression of the target gene by comparing the Ct values of the target gene and the internal reference gene (housekeeping gene) in different samples; absolute quantification requires the construction of a standard curve, and the absolute content of the target nucleic acid is calculated based on the standard curve and the Ct value of the sample.
Judgement and Reporting of Results
The results are judged according to the purpose of the experiment and the results of data analysis. For example, in pathogen detection, if the Ct value of the sample is lower than the set threshold of positivity, it will be judged as a positive result, indicating that the sample contains the target pathogen; in gene expression research, the change in the expression level of the target gene is described according to the results of relative quantification or absolute quantification.
The experimental results are collated into a report, including sample information, experimental methods, reaction conditions, data analysis methods and conclusions of the results, so as to facilitate subsequent research or clinical application.