Primer design and amplification efficiencies are crucial for reliability of quantitative PCR studies of caffeine biosynthetic N-methyltransferases in coffee.

Primers having suboptimal amplification efficiencies were shown to falsely represent fold change expression of the N-methyltransferases
gene family involved in caffeine biosynthesis in Coffea canephora. To study this phenomenon, the role of stability
of the internal reference gene, as well as the amplification efficiency correction of the primers was investigated. GAPDH and
Ubiquitin exhibited a good stability for studying the ontogeny of endosperm tissue, as well as the leaf transcriptome during
stress from salicylic acid, methyl jasmonate, PEG-mediated drought and sudden exposure to light. Ubiquitin manifested low
variation in Cq under all these stress regimes and in endosperm ontogeny with 30.1–30.9 in the best dataset and 28.8–30.9
in the most deviating dataset. It was observed that problems arising due to improper amplification efficiency of the target or
reference genes or both could lead to misinterpretation of gene expression levels. Quantitative RT-PCR performed at a suboptimal
efficiency of GAPDH reference gene at 1.68 led to the faulty interpretation of 2.007 folds upregulation by the 2−
ΔΔCt
method and 1.705 folds upregulation by Efficiency method for the first NMT (Xanthosine methyltransferase), which actually
is repressed during dark acclimatization of coffee plants. Efficiency correction improved the reliability of the expression data
and also indicated a downregulation of this gene by 0.485 folds and 0.474 folds using 2−
ΔΔCt and E method, respectively,
in concordance to earlier reports. Hence, efficiency correction of the primers having suboptimal efficiencies is an absolute
prerequisite for the accurate calculation of fold change using quantitative RT-PCR.