ArticleBMC genomics2015
Simple regression for correcting ΔCt bias in RT-qPCR low-density array data normalization.
Article in BMC genomics, 2015. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 9 papers, 1 of them a synthesis that pooled it.
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Who cites it
9 citing papers in PubMed, 1 synthesis or guideline pooled it.
- Replication Study:eLife · 2018Pooled it
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- Measuring and mitigating PCR bias in microbiota datasets.PLoS computational biology · 2021Article
- Testing the carotenoid-based sexual signalling mechanism by alteringProceedings. Biological sciences · 2020Article
- Transcriptomics in Toxicogenomics, Part II: Preprocessing and Differential Expression Analysis for High Quality Data.Nanomaterials (Basel, Switzerland) · 2020Review
- Toxicity Evaluation and Biomarker Selection with Validated Reference Gene in Embryonic Zebrafish Exposed to Mitoxantrone.International journal of molecular sciences · 2018Article
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Corrections and comments
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Authors and funding
14 authors.
Funding
Abstract
backgroundReverse transcription quantitative PCR (RT-qPCR) is considered the gold standard for quantifying relative gene expression. Normalization of RT-qPCR data is commonly achieved by subtracting the Ct values of the internal reference genes from the Ct values of the target genes to obtain ΔCt. ΔCt values are then used to derive ΔΔCt when compared to a control group or to conduct further statistical analysis.
resultsWe examined two rheumatoid arthritis RT-qPCR low density array datasets and found that this normalization method introduces substantial bias due to differences in PCR amplification efficiency among genes. This bias results in undesirable correlations between target genes and reference genes, which affect the estimation of fold changes and the tests for differentially expressed genes. Similar biases were also found in multiple public mRNA and miRNA RT-qPCR array datasets we analysed. We propose to regress the Ct values of the target genes onto those of the reference genes to obtain regression coefficients, which are then used to adjust the reference gene Ct values before calculating ΔCt.
conclusionsThe per-gene regression method effectively removes the ΔCt bias. This method can be applied to both low density RT-qPCR arrays and individual RT-qPCR assays.
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