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Also, ComBat and XPN give increased variability between replicates compared to standardisation in some cases, showing that cross-platform normalisation comes with a cost in terms of replicates.
Although all methods tested here provided similar global results and biological conclusions, the new direct random-primed cDNA labeling method provided slightly better correlation between replicates compared to the other methods and thus increased ability to find statistically significant differentially expressed genes.
The correlation between the qPCR and dPCR measurements, using the optimal template methylation range values, was also significant (R = 0.93; p < 0.0001; Figure 1E) however, analysis of the standard deviation values (Table 1 and Figure 1) revealed that, in the majority of cases, dPCR showed greater variability of measurement between replicates compared to qPCR.
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However, a certain level of sequence divergence between the two species could increase the random noise in the hybridization data and possibly result in a lower correlation between V. riparia replicates compared to V. vinifera replicates.
The 24K panel revealed less variation between both technical and extract replicates compared to the 1.5K panel.
For example, for the 4 h runs the overlap between the peptides identified using a linear gradient, and the ones found using an optimized one, ranged between 66% and 70% across the four replicates, compared to 74 82% when comparing any two replicates based on linear gradients.
The graph shows less dispersion between the filtered regions of the 12 technical replicates compared to the unfiltered regions.
The variance (standard error) was very small between the PCR replicates for a same biological sample (Real-Time experimental replicate) compared to the variance between the different biological replicates.
An inspection of the distributions of between replicate variances in our array data revealed that, indeed, D. sechellia showed significantly reduced between replicate variance compared to the other two species and the hybrids during the three earliest sampled times in our ontogenic interval (Kruskal-Wallis rank sum test, P < 2.2 × 10-16; Additional File 4).
For example, 40.6% of the total variation in reported expressions were attributed to replicate extractions, compared to 13.9% due to amplification/labelling and 10.8% between replicate hybridisations.
We also noted that Bmf-mediated cytochrome c release was much more variable between biological replicates compared with other peptides.
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