Normalization using ploidy and genomic DNA copy number allows absolute quantification of transcripts, proteins and metabolites in cells

• Main Authors: Matsui Minami, Takahashi Naoki, Obayashi Takeshi, Shimada Hiroshi, Sakamoto Atsushi
• Format: Article
• Language: English
• Published: BMC 2010-12-01
• Series: Plant Methods
• Online Access: http://www.plantmethods.com/content/6/1/29

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>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, <it>abc4</it>, 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 <it>RBC-L </it>and <it>RBC-S </it>(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 <it>RBC-L </it>and <it>RBC-S </it>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 × 10³transcripts of <it>RBC-L</it>, 9.9 × 10³transcripts of <it>RBC-S</it>, and 1.4 × 10⁶<it>18S rRNA</it>. We similarly analyzed the accumulation of RBC-L and LHCP (light-harvesting chlorophyll <it>a/b </it>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.</p> <p>Conclusion</p> <p>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.</p>