Direct cell lysis for single-cell gene expression profiling.
Artikel i vetenskaplig tidskrift, 2013

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.

cell lysis

RNA purification

direct lysis

DNA spike

RNA spike

single-cell biology

single-cell gene expression

real-time PCR

Författare

David Svec

Daniel Andersson

Göteborgs universitet

Milos Pekny

Göteborgs universitet

Robert Sjöback

Anders Ståhlberg

Göteborgs universitet

Frontiers in Oncology

2234-943X (ISSN)

Vol. 3 274-

Ämneskategorier

Cell- och molekylärbiologi

Medicinsk bioteknologi (med inriktning mot cellbiologi (inklusive stamcellsbiologi), molekylärbiologi, mikrobiologi, biokemi eller biofarmaci)

DOI

10.3389/fonc.2013.00274

PubMed

24224157