Q: What is multiplexing?
A: This is when the normalizer and the target gene are detected in the same well by assigning different dyes to each specific probe.
Q: How many genes can be detected in each well?
A: For optimal conditions 2 genes are the best, however, 3 or more can be done.
Q: Which dyes are best to multiplex?
A: We routinely multiplex FAM and VIC, however, other dyes can be multiplexed.
Q: Can all the housekeeping genes be used in multiplexing?
A: No, 18s is unable to be used in the same well as other targets due to its high abundance in samples. This causes it to inhibit the detection of the target genes.
Q: How do you make sure that there is no interaction between the normalizer and the target?
A: All the primers are tested before using them on the samples. The Ct or copy # values are compared between the primers when done alone and when multiplexed to make sure neither is inhibited.