title: Evaluation and validation of a robust single cell RNA-amplification protocol through transcriptional profiling of enriched lung cancer initiating cells.
abstract: BACKGROUND
  Although profiling of RNA in single cells has broadened our understanding of development, cancer biology and mechanisms of disease dissemination, it requires the development of reliable and flexible methods. Here we demonstrate that the EpiStem RNA-Amp™ methodology reproducibly generates microgram amounts of cDNA suitable for RNA-Seq, RT-qPCR arrays and Microarray analysis.
  
  
  RESULTS
  Initial experiments compared amplified cDNA generated by three commercial RNA-Amplification protocols (Miltenyi μMACS™ SuperAmp™, NuGEN Ovation® One-Direct System and EpiStem RNA-Amp™) applied to single cell equivalent levels of RNA (25-50 pg) using Affymetrix arrays. The EpiStem RNA-Amp™ kit exhibited the highest sensitivity and was therefore chosen for further testing. A comparison of Affymetrix array data from RNA-Amp™ cDNA generated from single MCF7 and MCF10A cells to reference controls of unamplified cDNA revealed a high degree of concordance. To assess the flexibility of the amplification system single cell RNA-Amp™ cDNA was also analysed using RNA-Seq and high-density qPCR, and showed strong cross-platform correlations. To exemplify the approach we used the system to analyse RNA profiles of small populations of rare cancer initiating cells (CICs) derived from a NSCLC patient-derived xenograft. RNA-Seq analysis was able to identify transcriptional differences in distinct subsets of CIC, with one group potentially enriched for metastasis formation. Pathway analysis revealed that the distinct transcriptional signatures demonstrated in the CIC subpopulations were significantly correlated with published stem-cell and epithelial-mesenchymal transition signatures.
  
  
  CONCLUSIONS
  The combined results confirm the sensitivity and flexibility of the RNA-Amp™ method and demonstrate the suitability of the approach for identifying clinically relevant signatures in rare, biologically important cell populations.
authors.affiliation: Nucleic Acid Biomarker Laboratory, Clinical & Experimental Pharmacology, CR-UK Manchester Institute, University of Manchester, Manchester M20 4BX, UK. Dominic.Rothwell@ics.manchester.ac.uk.
authors.forename: Dominic G
authors.initials: DG
authors.lastname: Rothwell
authors.forename: Yaoyong
authors.initials: Y
authors.lastname: Li
authors.forename: Mahmood
authors.initials: M
authors.lastname: Ayub
authors.forename: Catriona
authors.initials: C
authors.lastname: Tate
authors.forename: Gillian
authors.initials: G
authors.lastname: Newton
authors.forename: Yvonne
authors.initials: Y
authors.lastname: Hey
authors.forename: Louise
authors.initials: L
authors.lastname: Carter
authors.forename: Suzanne
authors.initials: S
authors.lastname: Faulkner
authors.forename: Massimo
authors.initials: M
authors.lastname: Moro
authors.forename: Stuart
authors.initials: S
authors.lastname: Pepper
authors.forename: Crispin
authors.initials: C
authors.lastname: Miller
authors.forename: Fiona
authors.initials: F
authors.lastname: Blackhall
authors.forename: Giulia
authors.initials: G
authors.lastname: Bertolini
authors.forename: Luca
authors.initials: L
authors.lastname: Roz
authors.forename: Caroline
authors.initials: C
authors.lastname: Dive
authors.forename: Ged
authors.initials: G
authors.lastname: Brady
mesh_terms: D045744:Cell Line, Tumor; D020869:Gene Expression Profiling; D006801:Humans; D008168:Lung / Q000473:pathology*; D014411:Neoplastic Stem Cells / Q000378:metabolism* / Q000473:pathology*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis; D060888:Real-Time Polymerase Chain Reaction; D015203:Reproducibility of Results; D017423:Sequence Analysis, RNA; D059010:Single-Cell Analysis / Q000379:methods*