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In these early days of whole-genome assays, efficiency of data collection may be enhanced, but the interpretation of findings still tends to be in terms of discrete gene loci, and the meaning of independent variations in the sequence.
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This maximizes assay efficiency and eliminates technical variability in both sample preparation and analysis.
Analysis of Cq and assay efficiency between control and target samples for the SPUD assay indicates the extent of matrix inhibition [ 16, 77].
Results were related to assay efficiency and GAPDH (GAPDH assay from Tataa Biocenter's Reference Gene Panel Human) and calculated according to comparative Ct method.
Different concentrations of perchlorate were tested with A. suillum grown anaerobically on acetate as the sole electron donor and perchlorate as the electron acceptor to determine assay efficiency.
Likewise, variations due to Taqman assay efficiency are likely to be minimised by performing RT-qPCR reactions for a panel of multiple standards.
Moreover, we verified that a comparable performance with respect to assay efficiency, precision, and accuracy can also be achieved in a routine molecular diagnostic laboratory.
For RT-qPCR measurements, calibration-curve-estimated copy number or fold changes should be reported rather than Cq, which is an arbitrary measure, and assay efficiency should ideally be taken into account.
Even though RT-qPCR is a method of choice, the reliability and reproducibility of experimental results for quantitative gene expression is dependent on the quality of RNA template and cDNA, primer specificity, assay efficiency, experimental conditions and rigorous analysis of the data using appropriate quality controls [ 41, 42].
Note that as for the TRAK2 immunoprecipitation assays, the efficiency of TRAK1 immunoprecipitations is not affected by the co-expression of the different KIF5A constructs (Fig. 5).
qPCR assay efficiency is shown in Supplementary Fig. 8.
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