title: Quantitative PCR analysis of DNA, RNAs, and proteins in the same single cell.
abstract: BACKGROUND
  The single cell represents the basic unit of all organisms. Most investigations have been performed on large cell populations, but understanding cell dynamics and heterogeneity requires single-cell analysis. Current methods for single-cell analysis generally can detect only one class of analytes.
  
  
  METHODS
  Reverse transcription and the proximity ligation assay were coupled with quantitative PCR and used to quantify any combination of DNA, mRNAs, microRNAs (miRNAs), noncoding RNAs (ncRNAs), and proteins from the same single cell. The method was used on transiently transfected human cells to determine the intracellular concentrations of plasmids, their transcribed mRNAs, translated proteins, and downstream RNA targets.
  
  
  RESULTS
  We developed a whole-cell lysis buffer to release unfractionated DNA, RNA, and proteins that would not degrade any detectable analyte or inhibit the assay. The dynamic range, analytical sensitivity, and specificity for quantifying DNA, mRNAs, miRNAs, ncRNAs, and proteins were shown to be accurate down to the single-cell level. Correlation studies revealed that the intracellular concentrations of plasmids and their transcribed mRNAs were correlated only moderately with translated protein concentrations (Spearman correlation coefficient, 0.37 and 0.31, respectively; P < 0.01). In addition, an ectopically expressed gene affected the correlations between analytes and this gene, which is related to gene regulation.
  
  
  CONCLUSIONS
  This method is compatible with most cell-sampling approaches, and generates output for the same parameter for all measured analytes, a feature facilitating comparative data analysis. This approach should open up new avenues in molecular diagnostics for detailed correlation studies of multiple and different classes of analytes at the single-cell level.
authors.affiliation: Department of Pathology, Sahlgrenska Cancer Center, University of Gothenburg, Gothenburg, Sweden. anders.stahlberg@gu.se
authors.forename: Anders
authors.initials: A
authors.lastname: Ståhlberg
authors.forename: Christer
authors.initials: C
authors.lastname: Thomsen
authors.forename: David
authors.initials: D
authors.lastname: Ruff
authors.forename: Pierre
authors.initials: P
authors.lastname: Åman
mesh_terms: D002021:Buffers; D045744:Cell Line, Tumor; D004247:DNA / Q000032:analysis*; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D006801:Humans; D035683:MicroRNAs / Q000032:analysis; D016133:Polymerase Chain Reaction / Q000379:methods*; D011506:Proteins / Q000032:analysis*; D012313:RNA / Q000032:analysis*; D012333:RNA, Messenger / Q000032:analysis; D022661:RNA, Untranslated / Q000032:analysis; D034702:RNA-Binding Protein FUS / Q000032:analysis / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics; D059010:Single-Cell Analysis
chemical_list: D002021:Buffers; D035683:MicroRNAs; D011506:Proteins; D012333:RNA, Messenger; D022661:RNA, Untranslated; D034702:RNA-Binding Protein FUS; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D012313:RNA; D004247:DNA