title: External calibration with Drosophila whole-cell spike-ins delivers absolute mRNA fold changes from human RNA-Seq and qPCR data.
abstract: Gene expression measurements are typically performed on a fixed-weight aliquot of RNA, which assumes that the total number of transcripts per cell stays nearly constant across all conditions. In cases where this assumption does not hold (e.g., when comparing cell types with different cell sizes) the expression data provide a distorted view of cellular events. Assuming constant numbers of total transcripts, increases in expression of some RNAs must be compensated for by decreases in expression of others. Therefore, we propose calibrating gene expression data to an external reference point, the number of cells in the sample, using whole-cell spike-ins. In a systematic dilution experiment, we mixed varying numbers of human cells with fixed numbers ofDrosophila melanogastercells and scaled the expression levels of the human genes relative to those of theDrosophilagenes. This approach restored the original gene expression ratios generated by the dilutions. We then usedDrosophilawhole-cell spike-ins to uncover non-symmetric gene expression changes, in this case much larger numbers of induced than repressed genes, under perturbations of the human cell line P493-6.Drosophilawhole-cell spike-ins are an experimentally and computationally easy and low-priced method to derive mRNA fold changes of absolute abundances from RNA sequencing (RNA-Seq) and quantitative real-time PCR (qPCR) data.
authors.affiliation: Statistical Bioinformatics, University of Regensburg, Regensburg, Germany.
authors.forename: Franziska
authors.initials: F
authors.lastname: Taruttis
authors.affiliation: Haematology and Oncology, University Medical Center Göttingen, Göttingen, Germany.
authors.forename: Maren
authors.initials: M
authors.lastname: Feist
authors.affiliation: Institute of Functional Genomics, University of Regensburg, Regensburg, Germany.
authors.forename: Phillip
authors.initials: P
authors.lastname: Schwarzfischer
authors.affiliation: Institute of Functional Genomics, University of Regensburg, Regensburg, Germany.
authors.forename: Wolfram
authors.initials: W
authors.lastname: Gronwald
authors.affiliation: Haematology and Oncology, University Medical Center Göttingen, Göttingen, Germany.
authors.forename: Dieter
authors.initials: D
authors.lastname: Kube
authors.affiliation: Statistical Bioinformatics, University of Regensburg, Regensburg, Germany.
authors.forename: Rainer
authors.initials: R
authors.lastname: Spang
authors.affiliation: Statistical Bioinformatics, University of Regensburg, Regensburg, Germany.
authors.forename: Julia C
authors.initials: JC
authors.lastname: Engelmann
mesh_terms: D000818:Animals; D002138:Calibration; D002460:Cell Line; D002478:Cells, Cultured; D004330:Drosophila; D020869:Gene Expression Profiling / Q000379:methods* / Q000592:standards; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D017423:Sequence Analysis, RNA / Q000379:methods*
chemical_list: D012333:RNA, Messenger
keywords: RNA sequencing (RNA-Seq); calibration; external reference; qPCR, gene expression; spike-in