Application of an RNA amplification method for reliable single-cell transcriptome analysis.
Diverse cell types have unique transcriptional signatures that are best interrogated at single-cell resolution. Here we describe a novel RNA amplification approach that allows for high fidelity gene profiling of individual cells. This technique significantly diminishes the problem of 3' bias, enabling detection of all regions of transcripts, including the recognition of mRNA with short or completely absent poly(A) tails, identification of noncoding RNAs, and discovery of the full array of splice isoforms from any given gene product. We assess this technique using statistical and bioinformatics analyses of microarray data to establish the limitations of the method. To demonstrate applicability, we profiled individual cells isolated from the mouse subventricular zone (SVZ)-a well-characterized, discrete yet highly heterogeneous neural structure involved in persistent neurogenesis. Importantly, this method revealed multiple splice variants of key germinal zone gene products within individual cells, as well as an unexpected coexpression of several mRNAs considered markers of distinct and separate SVZ cell types. These findings were independently confirmed using RNA-fluorescence in situ hybridization (RNA-FISH), contributing to the utility of this new technology that offers genomic and transcriptomic analysis of small numbers of dynamic and clinically relevant cells.
Department of Neurosurgery, College of Medicine, the Evelyn F. and William L. McKnight Brain Institute, University of Florida, Gainesville, FL.
Oleg
O
Suslov
Department of Neurosurgery, College of Medicine, the Evelyn F. and William L. McKnight Brain Institute, University of Florida, Gainesville, FL.|Human Biology Division, Fred Hutchinson Cancer Research Center, Seattle, WA.
Daniel J
DJ
Silver
Department of Neurosurgery, College of Medicine, the Evelyn F. and William L. McKnight Brain Institute, University of Florida, Gainesville, FL.|European Cancer Stem Cell Research Institute, Cardiff University, Cardiff, United Kingdom.
Florian A
FA
Siebzehnrubl
Biosearch Technologies, Petaluma, CA.
Arturo
A
Orjalo
Whitney Laboratory for Marine Biosciences, University of Florida, St. Augustine, FL.|Research Biomedical Informatics Division, Sidra Medical and Research Centre, Doha, Qatar.
Andrey
A
Ptitsyn
Department of Neurosurgery, College of Medicine, the Evelyn F. and William L. McKnight Brain Institute, University of Florida, Gainesville, FL.|Neuroscience and Aging Lab, Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University, Boston, MA.
Dennis A
DA
Steindler
D000071916:AC133 Antigen; D000818:Animals; D015703:Antigens, CD / Q000235:genetics; D045744:Cell Line, Tumor; D004268:DNA-Binding Proteins; D066246:ErbB Receptors / Q000235:genetics; D005136:Eye Proteins / Q000235:genetics; D020869:Gene Expression Profiling / Q000379:methods*; D005904:Glial Fibrillary Acidic Protein / Q000235:genetics; D006023:Glycoproteins / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics; D018398:Homeodomain Proteins / Q000235:genetics; D006801:Humans; D051798:Inhibitor of Differentiation Protein 1 / Q000235:genetics; D020547:Lateral Ventricles / Q000166:cytology; D008565:Membrane Proteins / Q000235:genetics; D008822:Mice, Transgenic; D009419:Nerve Tissue Proteins / Q000235:genetics; D009687:Nuclear Proteins / Q000235:genetics; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors / Q000235:genetics; D010455:Peptides / Q000235:genetics; D012313:RNA / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D012097:Repressor Proteins / Q000235:genetics; D059010:Single-Cell Analysis / Q000379:methods*
D000071916:AC133 Antigen; D015703:Antigens, CD; D004268:DNA-Binding Proteins; D005136:Eye Proteins; D005904:Glial Fibrillary Acidic Protein; D006023:Glycoproteins; D018398:Homeodomain Proteins; C497455:Idb1 protein, mouse; D051798:Inhibitor of Differentiation Protein 1; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; C492380:NeuN protein, mouse; D009687:Nuclear Proteins; C106293:Numb protein, mouse; D000071841:PAX6 Transcription Factor; C000605730:PAX6 protein, human; D051761:Paired Box Transcription Factors; C000605732:Pax6 protein, mouse; D010455:Peptides; D012097:Repressor Proteins; C528900:glial fibrillary astrocytic protein, mouse; D049452:Green Fluorescent Proteins; D012313:RNA; C541485:EGFR protein, mouse; D066246:ErbB Receptors
RNA amplification; SVZ; single-cell analysis; stem and progenitor cells