Direct cell lysis for single-cell gene expression profiling.
The interest to analyze single and few cell samples is rapidly increasing. Numerous extraction protocols to purify nucleic acids are available, but most of them compromise severely on yield to remove contaminants and are therefore not suitable for the analysis of samples containing small numbers of transcripts only. Here, we evaluate 17 direct cell lysis protocols for transcript yield and compatibility with downstream reverse transcription quantitative real-time PCR. Four endogenously expressed genes are assayed together with RNA and DNA spikes in the samples. We found bovine serum albumin (BSA) to be the best lysis agent, resulting in efficient cell lysis, high RNA stability, and enhanced reverse transcription efficiency. Furthermore, we found direct cell lysis with BSA superior to standard column based extraction methods, when analyzing from 1 up to 512 mammalian cells. In conclusion, direct cell lysis protocols based on BSA can be applied with most cell collection methods and are compatible with most analytical workflows to analyze single-cells as well as samples composed of small numbers of cells.
Institute of Biotechnology AS CR , Prague , Czech Republic ; TATAA Biocenter , Gothenburg , Sweden.
David
D
Svec
Daniel
D
Andersson
Milos
M
Pekny
Robert
R
Sjöback
Mikael
M
Kubista
Anders
A
Ståhlberg
DNA spike; RNA purification; RNA spike; cell lysis; direct lysis; real-time PCR; single-cell biology; single-cell gene expression