Abstract
Single-cell RNA sequencing (scRNA-seq) can unmask transcriptional heterogeneity facilitating the detection of rare subpopulations at unprecedented resolution. In response to challenges related to coverage and quantity of transcriptome analysis, the lack of unbiased and absolutely quantitative validation methods hampers further improvements. Digital PCR (dPCR) represents such a method as we could show that the inherent partitioning enhances molecular detections by increasing effective mRNA concentrations. We developed a scRT-ddPCR method and validated it using two breast cancer cell lines, MCF7 and BT-474, and bulk methods. ErbB2, a low-abundant transcript in MCF7 cells, suffers from dropouts in scRNA-seq and thus calculated fold changes are biased. Using our scRT-ddPCR, we could improve the detection of ErbB2 and based on the absolute counts obtained we could validate the scRNA-seq fold change. We think this workflow is a valuable addition to the single-cell transcriptomic research toolbox and could even become a new standard in fold change validation because of its reliability, ease of use and increased sensitivity.
Competing Interest Statement
J.S., E.S. and C.N. are employees of CYTENA GmbH, which produces the F.SIGHT single cell dispenser used in this study. T.L., T.G., P.K, and C.J. are employees of Actome GmbH, which develops the LBT lysis buffer used in this study and P.K., R.Z. and C.J. are shareholders of Actome GmbH. The remaining authors declare no competing interest.
Footnotes
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Abbreviations
- CI
- confidence interval,
- cl
- crude lysate,
- CV
- coefficient of variance,
- DE
- differential expression,
- DEG
- differentially expressed gene,
- dPCR
- digital PCR,
- FACS
- fluorescence-activated cell sorting,
- log2FC
- log2 of fold change between two conditions,
- RT-qPCR
- reverse transcription quantitative PCR,
- sc
- single-cell,
- scRNA-seq
- single-cell RNA sequencing,
- scRT-ddPCR
- single-cell reverse transcription droplet digital PCR,
- TPM
- Transcripts per kilobase million,
- UMAP
- uniform manifold approximation and projection,