Single cells are basic physiological and biological units that can function individually as well as in groups in tissues and organs. It is central to identify, characterize and profile single cells at molecular level to be able to distinguish different kinds, to understand their functions and determine how they interact with each other. During the last decade several technologies for single-cell profiling have been developed and used in various applications, revealing many novel findings. Quantitative PCR (qPCR) is one of the most developed methods for single-cell profiling that can be used to interrogate several analytes, including DNA, RNA and protein. Single-cell qPCR has the potential to become routine methodology but the technique is still challenging, as it involves several experimental steps and few molecules are handled. Here, we discuss technical aspects and provide recommendation for single-cell qPCR analysis. The workflow includes experimental design, sample preparation, single-cell collection, direct lysis, reverse transcription, preamplification, qPCR and data analysis. Detailed reporting and sharing of experimental details and data will promote further development and make validation studies possible. Efforts aiming to standardize single-cell qPCR open up means to move single-cell analysis from specialized research settings to standard research laboratories.

Sahlgrenska Cancer Center Department of Pathology and Genetics Institute of Biomedicine The Sahlgrenska Academy University of Gothenburg Box 425 40530 Gothenburg Sweden

TATAA Biocenter Odinsgatan 28 41103 Gothenburg Sweden; Laboratory of Gene Expression Institute of Biotechnology Czech Academy of Sciences Prumyslova 595 252 50 Vestec Prague Czech Republic

References provided by Crossref.org

Tutorial: Guidelines for Single-Cell RT-qPCR

Preamplification with dUTP and Cod UNG Enables Elimination of Contaminating Amplicons