title: Normalization using ploidy and genomic DNA copy number allows absolute quantification of transcripts, proteins and metabolites in cells.
abstract: BACKGROUND
  Quantification of transcripts, proteins, or metabolites is straightforward when the factor used to normalize these values remains constant between samples. However, normalization factors often vary among samples and thus must be developed for each new analytical method.
  
  
  RESULTS
  We demonstrate quantification of transcript and protein levels in Arabidopsis based on genomic DNA copy number. We extracted total nucleic acid from 3-week-old rosette leaves of wild-type Arabidopsis and the pale-green/dwarf mutant, abc4, and quantified the number of transcripts by quantitative reverse-transcription PCR using genomic DNA copy number and ploidy (as determined by cytometry) for normalization. Our data indicated that normalization using genes commonly employed as references resulted in inaccuracies in transcript levels of the genes RBC-L and RBC-S (encoding the large and small subunits, respectively, of ribulose 1,5-bisphosphate carboxylase/oxygenase) in wild type and mutant. Normalization using genomic DNA copy number and ploidy, however, appropriately showed that the RBC-L and RBC-S transcript levels per cell in the mutant were significantly lower than that in wild type. Furthermore, quantification revealed that a cell of a 3-week-old wild-type Arabidopsis rosette leaf had an average of 7.5 × 103 transcripts of RBC-L, 9.9 × 103 transcripts of RBC-S, and 1.4 × 106 18S rRNA. We similarly analyzed the accumulation of RBC-L and LHCP (light-harvesting chlorophyll a/b protein) in wild type and mutant based on ploidy and genomic DNA copy number that was determined by direct quantitative PCR analysis of extracts using a DNA polymerase tolerant to a wide range of common PCR inhibitors. Furthermore, we estimated the number of RBC-L molecules (2.63 × 108) and chlorophyll molecules (1.85 × 109) in each cell in 3-week-old wild-type rosette leaves; these values had relatively low coefficients of variation, underscoring the reliability of our method.
  
  
  CONCLUSIONS
  Genomic DNA copy number and ploidy are useful as general normalization factors, providing an easy method for determining the number of transcripts, proteins, and metabolites in a cell.
authors.affiliation: Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, 1-3-1, Kagamiyama, Higashi-Hiroshima, 739-8526, Japan. hshimada@hiroshima-u.ac.jp.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Shimada
authors.forename: Takeshi
authors.initials: T
authors.lastname: Obayashi
authors.forename: Naoki
authors.initials: N
authors.lastname: Takahashi
authors.forename: Minami
authors.initials: M
authors.lastname: Matsui
authors.forename: Atsushi
authors.initials: A
authors.lastname: Sakamoto