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Proteins identified in two or more technical replicates per category were kept for further analysis.
We also maintain the opinion [ 6] that the MAQC study should be continued to generate more technical replicates for a more accurate assessment of the random noise component.
We speculate that the inclusion of more technical replicates, or even biological replicates, may contribute to decrease the number of false-positive genes.
This error margin may be reduced by running more technical replicates (not tested); however, for our analyses we deemed this not necessary as the main aim was to select plants with a unique insertion as efficiently as possible.
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For a certain biological condition, one or more wells representing technical replicates can be selected.
Myh isoform analysis and PCA revealed that biological replicates were in general more different than technical replicates (Fig 2D, see Supplementary Fig S3A for comparison) and were in some cases quite distinct at the proteome level (Fig 2D and E, Supplementary Fig S3D).
Technical replicates are used for the qPCR data, and where biological replicates are shown there is clearly more variation than between technical replicates, but interpretations of the result are not affected by this level of uncertainty.
Data reliability was assessed by checking Pearson correlation coefficient C among samples: technical replicates resulted more correlated among themselves (C>0.96).
While, increasing the number of biological replicates increases the precision and generalizability of a study more than increasing the number of technical replicates; for studies where low abundant mRNAs are the focus, increasing technical replication may also be important.
Proteins identified in more than one technical replicate in a single category and not in any category were also considered for functional analysis as unique identifications.
Assays were repeated three or more times with three to six technical replicates per cell line each time.
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