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Nat Protoc. 2015 Jul;10(7):974-84. doi: 10.1038/nprot.2015.058. Epub 2015 Jun 4.

Single prokaryotic cell isolation and total transcript amplification protocol for transcriptomic analysis.

Author information

1
Department of Microbiology, University of Hawaii at Manoa, Honolulu, Hawaii, USA.
2
Department of Molecular Biosciences and Bioengineering, University of Hawaii at Manoa, Honolulu, Hawaii, USA.
3
1] Department of Molecular Biosciences and Bioengineering, University of Hawaii at Manoa, Honolulu, Hawaii, USA. [2] Present address: Department of Infectious Diseases and Pathology, University of Florida, Gainesville, Florida, USA.
4
1] Department of Microbiology, University of Hawaii at Manoa, Honolulu, Hawaii, USA. [2] Department of Molecular Biosciences and Bioengineering, University of Hawaii at Manoa, Honolulu, Hawaii, USA.

Abstract

Until recently, transcriptome analyses of single cells have been confined to eukaryotes. The information obtained from single-cell transcripts can provide detailed insight into spatiotemporal gene expression, and it could be even more valuable if expanded to prokaryotic cells. Transcriptome analysis of single prokaryotic cells is a recently developed and powerful tool. Here we describe a procedure that allows amplification of the total transcript of a single prokaryotic cell for in-depth analysis. This is performed by using a laser-capture microdissection instrument for single-cell isolation, followed by reverse transcription via Moloney murine leukemia virus, degradation of chromosomal DNA with McrBC and DpnI restriction enzymes, single-stranded cDNA (ss-cDNA) ligation using T4 polynucleotide kinase and CircLigase, and polymerization of ss-cDNA to double-stranded cDNA (ds-cDNA) by Φ29 polymerase. This procedure takes ∼5 d, and sufficient amounts of ds-cDNA can be obtained from single-cell RNA template for further microarray analysis.

PMID:
26042386
PMCID:
PMC4494743
DOI:
10.1038/nprot.2015.058
[Indexed for MEDLINE]
Free PMC Article

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