I want to detect the T-cell receptor excision circles (TRECs) in PBMC, but there is no standard test method in the published literature. The more used method is the quantitative PCR with probe and primers, and a standard curve is made with TREC plasmid. At the same time, RAG2 or GAPDH or β-actin is used as an internal reference.

However, I still have two questions that I would like to seek help and answers for: (1) Why do we need an internal reference gene when the TREC curve drawn from the standard is used for absolute quantification? (2) We want to make a RAG2 standard product for absolute quantification of cell numbers, and have found the upstream and downstream primers, but how can we determine the sequence of the RAG2 standard product based on its primers?