title: Validation of high-throughput single cell analysis methodology.
abstract: High-throughput quantitative polymerase chain reaction (qPCR) approaches enable profiling of multiple genes in single cells, bringing new insights to complex biological processes and offering opportunities for single cell-based monitoring of cancer cells and stem cell-based therapies. However, workflows with well-defined sources of variation are required for clinical diagnostics and testing of tissue-engineered products. In a study of neural stem cell lines, we investigated the performance of lysis, reverse transcription (RT), preamplification (PA), and nanofluidic qPCR steps at the single cell level in terms of efficiency, precision, and limit of detection. We compared protocols using a separate lysis buffer with cell capture directly in RT-PA reagent. The two methods were found to have similar lysis efficiencies, whereas the direct RT-PA approach showed improved precision. Digital PCR was used to relate preamplified template copy numbers to Cq values and reveal where low-quality signals may affect the analysis. We investigated the impact of calibration and data normalization strategies as a means of minimizing the impact of inter-experimental variation on gene expression values and found that both approaches can improve data comparability. This study provides validation and guidance for the application of high-throughput qPCR workflows for gene expression profiling of single cells.
authors.affiliation: LGC, Teddington, Middlesex TW11 0LY, UK. Electronic address: alison.devonshire@lgcgroup.com.
authors.forename: Alison S
authors.initials: AS
authors.lastname: Devonshire
authors.affiliation: LGC, Teddington, Middlesex TW11 0LY, UK.
authors.forename: Marc-Olivier
authors.initials: MO
authors.lastname: Baradez
authors.affiliation: LGC, Teddington, Middlesex TW11 0LY, UK.
authors.forename: Gary
authors.initials: G
authors.lastname: Morley
authors.affiliation: LGC, Teddington, Middlesex TW11 0LY, UK.
authors.forename: Damian
authors.initials: D
authors.lastname: Marshall
authors.affiliation: LGC, Teddington, Middlesex TW11 0LY, UK.
authors.forename: Carole A
authors.initials: CA
authors.lastname: Foy
mesh_terms: D002138:Calibration; D002460:Cell Line; D057230:Limit of Detection; D036103:Nanotechnology; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000235:genetics; D048348:Reverse Transcription; D059010:Single-Cell Analysis / Q000379:methods*
chemical_list: D012333:RNA, Messenger
keywords: Digital PCR; Gene expression; Normalization; RT–qPCR; Single cell; mRNA