Please use this identifier to cite or link to this item: http://hdl.handle.net/10125/40214

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

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dc.contributor.author Kang, Yun
dc.contributor.author McMillan, Ian
dc.contributor.author Norris, Michael H.
dc.contributor.author Hoang, Tung T.
dc.date.accessioned 2016-04-30T00:21:04Z
dc.date.available 2016-04-30T00:21:04Z
dc.date.issued 2015-07
dc.identifier.uri http://hdl.handle.net/10125/40214
dc.description.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 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 utilizing 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, ss-cDNA ligation using T4 polynucleotide kinase and CircLigase, and polymerization of ss-cDNA to ds-cDNA by ϕ 29 polymerase. This procedure takes ~5 days, and sufficient amounts of ds-cDNA can be obtained from single cell RNA template for further microarray analysis.
dc.format.extent 22
dc.language.iso en-US
dc.relation.uri http://www.nature.com/nprot/journal/v10/n7/full/nprot.2015.058.html
dc.relation.uri http://www.ncbi.nlm.nih.gov/pubmed/?term=%22Single+prokaryotic+cell+isolation+and+total+transcript+amplification+protocol+for+transcriptomic+analysis%22
dc.title Single prokaryotic cell isolation and total transcript amplification protocol for transcriptomic analysis
dc.type Article
dc.type.dcmi Text
dc.identifier.doi 10.1038/nprot.2015.058
Appears in Collections: Department of Microbiology Faculty & Researcher Works


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