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

Abstract

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 sub-optimal 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.

Assessment of Mathematical Approaches for the Estimation and Comparison of Efficiency in qPCR Assays for a Prokaryotic Model

“…Other experimental conditions can impact qPCR reactions and subsequent analyses, including primer design [32], template quality [25], enzymes (DNA polymerase types and conditions) [33], other reaction components [24], cycling conditions [34], sample handling and pipetting errors, reference genes for normalization [35], and others. Jointly, these parameters will be considered as part of further assays using bacterial models in a more biological context, including the quantification of determinants that we have previously found in the perturbome [17], the SOS response [36], or the response to ciprofloxacin [18] in P. aeruginosa AG1.…”

Considerations and Suggestions for the Reliable Analysis of miRNA in Plasma Using qRT-PCR

“…Therefore, in addition to exogenous miRNA, appropriate reference genes are needed to normalize extracted endogenous miRNA levels, and reference gene selection must be prioritized because some reference genes can differ depending on the sample condition and type. With normalization, the evaluation of amplification efficiency is also crucial for the reliability of qRT-PCR studies [ 37 , 73 , 74 ]. Sreedharan et al demonstrated improvements in the reliability of expression data through primer-dependent improvements in amplification efficiency [ 73 ].…”

“…With normalization, the evaluation of amplification efficiency is also crucial for the reliability of qRT-PCR studies [ 37 , 73 , 74 ]. Sreedharan et al demonstrated improvements in the reliability of expression data through primer-dependent improvements in amplification efficiency [ 73 ]. Variations in primer concentration and annealing temperature, as well as primer design, can affect amplification efficiency and consequently affect the reliability of expression data.…”

Genome-wide identification and expression analysis of the GRAS gene family under abiotic stresses in wheat (Triticum aestivum L.)

“…The primers were designed using the PrimerBlast tool 59 , with a final product size of approximately 150 base pairs (Supplementary Table S1 ). The primer efficiency was calculated by using the formula: E = 10 (-1/slope) 60 . The thermal cycling conditions for qRT-PCR was: 95 °C for 5 min, then 40 cycles of 94 °C for 15 s, 55 °C for 30 s and 72 °C for 30 s, and a final melting curve step from 65 °C to 95 °C with a ramp speed of 0.5 °C per 5 s. Actin, a housekeeping gene, was used as an internal control 61 (F: 5′-GGAGAAGCTCGCTTACGTG-3′ & R: 5′-GGGCACCTGAACCTTTCTGAA-3′).…”