Will repeated freeze-thaw cycles after dissolving primer and probe dry powder affect the experimental results?

The number of aliquots mainly depends on your weekly usage. Generally, it is recommended to aliquot the stock solution at 10-20 μL per tube, with no more than 1-2 freeze-thaw cycles; for working solution, each tube should contain enough for one use or one day, commonly 10 μM, 20-50 μL. For long-term storage, probes should preferably be stored at -80°C, while primers can usually be stored at -20°C; if -80°C is not convenient, -20°C storage should also be placed in the frost-free area of a frost-free freezer to avoid repeated freezing. TE pH 8.0 is more stable than pure water and helps avoid pH changes caused by repeated freeze-thaw cycles. Probes must be protected from light, using brown tubes or aluminum foil wrapping, especially fluorescent groups such as FAM and HEX, which will quench upon light exposure. Use low-binding tubes during aliquoting to reduce wall adsorption loss.

Repeated freeze-thaw cycles three or four times can indeed cause Ct values to drift, especially for probes. For lyophilized powder, centrifuge first, dissolve in TE or Tris-HCl to a 100 μM stock solution, then prepare a 10 μM working solution. Aliquot the working solution according to the amount needed per experiment, for example 20-50 μL per tube. Keep one commonly used tube at 4°C for short-term use, and store the rest at -20°C. Do not repeatedly freeze-thaw the stock solution, and do not take the entire tube out of the freezer, thaw it to room temperature, and then put it back. Probes carry fluorescent and quencher groups, and repeated freeze-thaw cycles may increase fragmentation and free fluorophores, causing an elevated baseline or a shift of Ct to later cycles. If the working solution has already been freeze-thawed multiple times, it is best to discard it, aliquot a new tube, and verify with standards.

High Ct variability isn't always caused by freeze-thaw cycles—you also need to check both the instrument and the reagents. I recommend comparing on the same plate: newly aliquoted probe vs. probe that has undergone three to four freeze-thaw cycles, run a gradient with the same template, and look at the Ct difference; at the same time, check whether the ROX reference and baseline are normal. If the problem is probe degradation, typically the NTC background is elevated, and low-copy sample Ct delay is more pronounced. For long-term storage, -80 is recommended; repeated freeze-thaw should be avoided, and during handling, centrifuge first before opening the cap. For critical projects, I make a habit of aliquoting into small volumes upon arrival and keeping one validation batch specifically for batch-to-batch comparison. If Ct fluctuation exceeds 0.5-1, first switch to a new aliquot tube and rerun, then consider re-synthesis.