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

Hiroshi Shimada,Atsushi Sakamoto,Minami Matsui,Naoki Takahashi,Takeshi Obayashi

doi:10.1186/1746-4811-6-29

Hiroshi Shimada, Atsushi Sakamoto + Show 3 more

Open Access

https://doi.org/10.1186/1746-4811-6-29

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Abstract

BackgroundQuantification 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.ResultsWe 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.ConclusionGenomic 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.

Highlights

Quantification of transcripts, proteins, or metabolites is straightforward when the factor used to normalize these values remains constant between samples
Because rRNA comprises a large proportion of total RNA in the cell, transcript quantification based on total amounts of RNA or mRNA in one cell type may not accurately reflect the transcript levels in other cell types
Quantification by Coomassie Brilliant Blue (CBB) staining of samples subjected to SDS-PAGE or by immunoblotting based on total input protein revealed similar levels of both RBC-L and RBC-S between wild type and abc4 and that the mutant had a slightly reduced level of LHCP [9]. To address these potentially confounding factors in quantitative analysis, we developed methods to quantify transcript, protein, and metabolite levels based on genomic DNA copy number and ploidy using A. thaliana wild type and abc4

Summary

Introduction

Quantification of transcripts, proteins, or metabolites is straightforward when the factor used to normalize these values remains constant between samples. Normalization factors often vary among samples and must be developed for each new analytical method. Proteins, and metabolites are usually quantified relative to the value for a known, constitutively expressed cellular factor. Quantification of transcripts using northern hybridization is based on total amounts of RNA or mRNA. Quantification of transcripts using RT-PCR analysis, including real-time RT-PCR, is based on the expression level of a reference gene [1,2,3,4], and a DNA array detects may vary widely depending on the cell population [7,8]. The precision of quantitative (q)RT-PCR depends on accurate transcript normalization using constitutively expressed genes. Total protein, fresh weight, dry weight, or culture volume may vary between samples

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