It is generally safe, provided that the plate is tightly sealed (ideally with optical adhesive film or caps) to prevent evaporation and contamination. And avoid repeated freeze-thaw cycles, which can degrade RNA and enzymes. When ready to run, thaw the plate on ice, spin it down briefly, and proceed directly with thermal cycling.

I personally wouldn't trust it, but haven't tested since the reagents are so expensive. Check the storage instructions for the kit to see if -20*C is OK or not.

If your data look "odd", the first thing you'd do is repeat.

Sara Benchikh Yes, it's generally safe to store a pre-assembled one-step qRT-PCR plate at –20 °C before running, as long as the plate is well sealed to prevent evaporation, protected from light (if using fluorescent dyes), and freeze-thaw cycles are minimized to preserve enzyme and RNA integrity. Thaw gently before use, and ensure all components are compatible with freezing—always follow the manufacturer’s guidelines for best results.