In what way could the reaction work better? Stronger product amount or cleaner product without primer dimer? How much dna are you adding to each reaction and how many cycles are you running and are you getting any primer dimer even faintly. 50ul is a large volume and may be slow to reach both denaturation and annealing teperatures so I would run 20 or 25ul reactions which reach temperatures quicker and possibly reduce times at annealing and extension since your amplimers are very small

Sorry, I didn’t phrase it correctly, I still haven't tried the multiplex PCR with both primer pairs, I've only done two separate reactions with each pair of primers adding 1ul of cDNA at 800ng/ul, since I am still uncertain on how to addapt the mix for multiplex PCR, but I do want to optimize for cleaner bands since I am interested in comparing band intensity between 2 different conditions.

Thanks so much for the tips, I already lowered extension time compared to the standard protocol, but I will also try to lower the final volume scaling down the components accordingly.

yes, do like you said. Your design seems fine, it should work. then run your PCR products on higly concentrated agarose gel to be able to fully distinguish the two products.

PS: Is there a specific reason why you want to use Phusion taq? If no, i would even try with standard Taq.

It should work with your changes but I think that 800ng is too much cDNA which does have a smaller size than genomic dna. You may want to run some dilutions (1/2 1/4 1/8 and 1/16) of the amount you are using as too much dna can often lead to failed pcr or increased non specific bands. Also a no template control is a good idea to see if primer dimer or contamination with previously amplified product is an issue