1. The library linker sequence is incorrect. Check if the library end sequence matches the primer sequence provided by the kit. 2. The dilution is too high. Reduce the dilution factor and repeat the experiment. 3. Library degradation. The library should be diluted now, and the diluted library should be placed on ice for use.

1. The Ct value of the standard product affected by pollution is not discarded. 2. All reagents should be fully thawed and thoroughly mixed before use. 3. Instrument related issues. Verify that the ROX Reference Dye used matches the quantitation instrument. 4. Poor pipetting accuracy.