 Solution

A housekeeping Ct is unnecessary for the within-sample short:long ratio, because a common reference cancels. With matched doubling efficiencies,However, a validated reference such as TATA-binding protein transcript is needed when comparing the abundance of either isoform separately between samples while correcting for input, RNA recovery and reverse transcription. Reference stability must be tested under the differentiation conditions. Neither reference normalization nor cancellation removes the need to validate the two isoform assays' efficiencies and response calibration.