Table 1.

Key modifications required for multiplex long amplicon droplet digital PCR (ddPCR) assays of alternatively-spliced transcripts.

	Key Assay Design Features
1.	Primers and probes designed with high melting temperatures (≥60 °C).
2.	Probe design critical—with splice site location close to 5’ end of probe where possible.
3.	Long extension times (4–6 min) with high cycle numbers (50 cycles).
4.	Two probes to one transcript can be used to allow detection of >2 transcripts in one reaction, if only two fluorophore channels available.