Mitigation of the effect of variability in digital PCR assays through use of duplexed reference assays for normalization.

ABSTRACT: Digital PCR has been promoted as a technique for obtaining absolute measures of the amount of nucleic acid target sequence in a sample, but still lacks standardization in data reporting. The initial method of representing data as copies per microliter produced inconsistent results and made inter-assay comparisons difficult. Normalizing copies to amount of nucleic acid gives more uniform results, but factors influencing the effective concentration of nucleic acid in the final digital PCR assay must be considered. Using droplet digital PCR and previously validated reference genes duplexed with target genes, a method of normalization was developed to estimate the amount of input nucleic acid in individual assays, subsequently reporting the number of copies of target gene relative to this amou

SUBMITTER: Coulter SJ 

PROVIDER: S-EPMC6424105 | biostudies-literature | 2018 Aug

REPOSITORIES: biostudies-literature