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15 Mean crossing point value of β-globin real time polymerase chain reaction assay was 23.84, standard deviation 0.95.
The expression ratios are calculated on the basis of the mean crossing point (CP) values for reference and target genes [ 10].
mRNA concentrations, based on mean crossing point (CP) values, were normalized against BCR mRNA level and expressed relative to a calibrator sample as previously described [ 16, 19].
Two groups (controls and EpCAM-positive cells) were compared based on their mean crossing point deviation for significance by a randomization test.
PCR efficiencies (E) were measured for each locus, and the fold-enrichment was calculated using the difference between the mean crossing point (CP) values reported between the precapture and the postcapture matrices (∆CP), with: fold enrichment = E∆CP.
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(a) Mean of crossing point values calculated using two replicates of each point in 3 runs.
Variation in each gene measurement was assessed using the difference between the mean calibrator crossing point (CP) and each sample replicate CP (ΔCP).
It means the crossing points of the 5 nonzero residual surfaces expanded by the 5 noisy data sets are quite close to the true parameter point.
The Bestkeeper algorithm measures the geometric mean of reference gene crossing point values, to determine the optimal reference gene for use in a samples set.
Real time PCR was performed in triplicate, and the mean for the crossing points of triplicates was used in subsequent calculations.
In normal, surrounding breast tissue, the mean value of crossing points (cycle threshold) was 34,02 whereas in the tumour tissue samples it was 36,22 (indicating low levels of CLDN1 mRNA).
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