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This graph demonstrates that plates from the same patient give consistent raw mean CT values prior to reference normalization.
Because the earlier biases differ from dataset to dataset, the reference normalization is performed as the final normalization step.
Reference normalization: We divided each experimental profile from the aneroid strains by its associated (via replicate) euploid control profile.
After data quality control and reference normalization, approximately 19% of the cancer related genes tested in this study were found to have a significant (p < 0.05) correlation with Recurrence Free Interval (RFI) by univariate regression analysis [ 10].
The Cq value for the reference normalization factor was calculated by taking the geometric mean of the four P. griseofulvum reference genes: the ribosomal 28S RNA, the 37S ribosomal protein (PGRI_092740), the beta-tubulin (PGRI_052690), and the histone H3 (PGRI_044770)).
The seven cancer-related genes (BGN, MYC, FAP, GADD45B, INHBA, MK167 and MYBL2) and five reference normalization genes (ATP5E, GPX1, PGK1, VDAC2 and UBB) were selected from 761 genes, based on the results of four development studies which included over 1800 patients [ 6].
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The geometric mean of expression of the two reference genes (normalization factor) was used for normalization.
For SNP data, the normal gastric samples were used as the reference for normalization.
Data pre-processing was performed using Microsoft Excel 2007 and included efficiencies and reference gene normalization.
Cycle of threshold (Ct) was determined from the triplicate experiments using the GAPDH gene transcription as reference for normalization.
Our treatment of reference in normalization is somewhat different from existing methods, and correspondingly we use this group median polishing summarization to take into account the reference effect.
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