Q1: Why no Ct value appears in the final result?
A1:
1) The steps of collecting the fluorescence signal are wrong. Check the program settings, two-step amplification collects signals in the annealing extension step, and three-step amplification collects signals in the extension stage.
2) Template or ROX usage error. When the ROX concentration is too low or too high, the Ct value may be displayed as Unknown or blank, select the appropriate ROX according to the instrument type.
3) Primer or template degradation. PAGE electrophoresis to check the integrity of the primers, agarose gel electrophoresis to check the quality and integrity of the template DNA or RNA, it is recommended to reverse-transcribe the RNA template to obtain cDNA, and the cDNA should be used as soon as possible.
4) The amount of template is insufficient. Appropriately increase the amount of template or re-prepare high-concentration template.
Q2: Why is the experiment reproducible?
A2:
1) There is an error in adding the sample. Use a pipette with better performance, prepare the premix and then dispense it to reduce pipetting errors.
2) The temperature control of different positions of the fluorescence quantitative PCR instrument is inconsistent. Periodically calibrate the instrument.
3) The template concentration is low or the copy number is low. Appropriately increase the template usage.
Q3: Why is the shape of the amplification curve abnormal?
A3:
1) The amplification curve is not smooth. If the signal is too weak, it will be generated after system correction, and the experiment can be repeated by increasing the template concentration.
2) The amplification curve breaks and drops sharply. If the template concentration is high or there are air bubbles in the reaction tube, reduce the baseline end point (CT value -4) and re-analyze the data, or centrifuge and carefully check whether there are air bubbles in the reaction tube before loading.
3) Incorrect channel selection when analyzing data. If ROX is not added to the system, but the ROX option is selected in the instrument settings during analysis, appropriate analysis conditions should be selected according to the specific operating system.
Q4: Why the same reagent produces different curves on different instruments?
A4:
Analyze the amplification efficiency, sensitivity and specificity. If the amplification efficiency is between 90% and 110%, and all of them are specific amplification, the data can be analyzed and used.
