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Technical aspects and recommendations for single-cell qPCR.

Review article
Authors Anders Ståhlberg
Mikael Kubista
Published in Molecular aspects of medicine
Volume 59
Pages 28-35
ISSN 1872-9452
Publication year 2018
Published at Sahlgrenska Cancer Center
Institute of Biomedicine, Department of Pathology
Pages 28-35
Language en
Links dx.doi.org/10.1016/j.mam.2017.07.00...
www.ncbi.nlm.nih.gov/entrez/query.f...
Subject categories Cell and Molecular Biology

Abstract

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.

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