title: Single prokaryotic cell isolation and total transcript amplification protocol for transcriptomic analysis.
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.
authors.affiliation: Department of Microbiology, University of Hawaii at Manoa, Honolulu, Hawaii, USA.
authors.forename: Yun
authors.initials: Y
authors.lastname: Kang
authors.affiliation: Department of Molecular Biosciences and Bioengineering, University of Hawaii at Manoa, Honolulu, Hawaii, USA.
authors.forename: Ian
authors.initials: I
authors.lastname: McMillan
authors.affiliation: 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.
authors.forename: Michael H
authors.initials: MH
authors.lastname: Norris
authors.affiliation: 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.
authors.forename: Tung T
authors.initials: TT
authors.lastname: Hoang
mesh_terms: D000818:Animals; D001419:Bacteria / Q000235:genetics / Q000302:isolation & purification; D002469:Cell Separation / Q000379:methods; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D020869:Gene Expression Profiling / Q000379:methods*; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D061346:Laser Capture Microdissection / Q000379:methods; D011387:Prokaryotic Cells / Q000166:cytology* / Q000378:metabolism*
chemical_list: D004269:DNA, Bacterial; D018076:DNA, Complementary