Normalization using ploidy and genomic DNA copy number allows absolute quantification of transcripts, proteins and metabolites in cells
© Shimada et al; licensee BioMed Central Ltd. 2010
Received: 1 October 2010
Accepted: 29 December 2010
Published: 29 December 2010
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.
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 × 10618S 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.
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.
Cellular levels of transcripts, 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 [1–4], and a DNA array detects relative levels of transcripts [5, 6]. Protein levels are typically quantified by Coomassie Brilliant Blue (CBB) staining of samples subjected to SDS-PAGE, by two-dimensional difference gel electrophoresis for proteome analysis, by immunoblotting, or by enzyme-linked immunosorbent assay relative to the weight of total protein, fresh weight, dry weight or culture volume. Metabolites are often quantified based on the weight of total protein, fresh weight or dry weight. Such quantification methods are useful when the normalization factor does not vary among samples. Between tissues, however, the transcriptional activity may differ, and the ratio between mRNA and rRNA may vary widely depending on the cell population [7, 8]. 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. The precision of quantitative (q)RT-PCR depends on accurate transcript normalization using constitutively expressed genes. Statistical algorithms have been developed to help validate reference genes [3, 4]; prior to analysis, however, it is difficult to know which reference gene is consistently expressed among the samples, such as when a novel mutant or treatment analysis is under consideration. Similarly, total protein, fresh weight, dry weight, or culture volume may vary between samples.
In Arabidopsis thaliana, abc4 is a mutant of the phylloquinone biosynthesis gene and exhibits the dwarf and pale-green phenotype . The mutant has fewer chloroplasts than wild type, and the intercellular space is also larger . Northern hybridization using total RNA revealed that the RBC-L (Rubisco large subunit) and RBC-S (Rubisco small subunit) transcript levels are significantly elevated in the abc4 mutant, whereas the LHCP (light-harvesting chlorophyll a/b protein) transcript level is almost the same as in the wild type . Quantification by 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 . 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.
Results and Discussion
Analysis of genomic DNA copy number per cell
Transcript accumulation normalized to genomic DNA copy number and ploidy
Northern hybridization using total RNA revealed that the RBC-L and RBC-S transcript levels were significantly elevated in abc4. Note that qRT-PCR normalized to the genes often used as references may provide misleading results (Figure 2A). However, qRT-PCR using genomic DNA copy number and the mean ploidy as the reference can provide more accurate information on the level of transcripts per cell.
Quantification of transcript number per cell
Each cell in the wild-type rosette leaf had an average of 7.5 × 103RBC-L transcripts, 9.9 × 103RBC-S transcripts, and 1.4 × 10618S rRNA transcripts (see additional file 4). The number of PPR/At1g62930 (pentatricopeptide repeat-containing protein gene) transcripts per cell was much lower (0.17) (Figure 2C; also see additional file 4), indicating that there is less than one PPR transcript per cell. PPR was not transcribed in all cells of wild-type rosette leaves. The rosette leaf contains various cell types (e.g., mesophyll cells, epidermal cells, guard cells and vascular tissue cells), and PPR transcription may be cell type specific. Of course, any gene with less than one transcript per cell should not be used as a reference for quantification of qRT-PCR data.
Protein accumulation normalized to genomic DNA copy number and ploidy
We next quantified protein expression levels normalized to genomic DNA copy number and ploidy established by qPCR using plant extracts. To extract total protein, the plants were homogenized in an extraction buffer containing 10 mM EDTA and 1.0% SDS, both of which inhibit DNase (see Methods) . Because the KAPA2G Robust HotStart DNA polymerase (Kapa Biosystems) used in this assay is tolerant to a wide range of common PCR inhibitors (e.g., salts and SDS), the protein samples were used without further purification as the qPCR template. Protein samples from equivalent numbers of wild type and mutant cells were then subjected to SDS-PAGE; for this analysis the volume of protein sample from the mutant (V mutant ) was applied. , where V wild is the applied volume of protein sample from wild type, R is the relative genomic DNA copy number of the protein sample from the mutant to that of wild type, and G mutant and G wild are the genomic DNA copy number per cell (i.e., mean ploidy) of the abc4 and wild-type plants, respectively (see Methods and additional file 5). The amounts of RBC-L and LHCP in abc4 were significantly lower than in the wild type based on genomic DNA copy number (Figure 3A lanes 7 and 8, and Figure 3B).
Quantification of protein and metabolite number per cell
We next analyzed the number of genomic DNA molecules and RBC-L molecules per cell in protein samples of 3-week-old wild-type rosette leaves. One microliter of a 1:40 dilution of wild-type protein extract and 1 μl of plasmid harboring a DNA fragment amplified by PCR (between 2.88 × 102 and 2.88 × 109 molecules, or 0 molecules) were added to the PCR reactions. This mixing procedure was done because the amplification efficiency of the qPCR differed depending on whether the protein extract or the purified plasmid was used as template (efficiency = 0.665 or 0.890, respectively). Therefore, we could not extrapolate the genomic DNA copy number in the protein samples using the standard curve created with the purified plasmid. Thus, to estimate genomic DNA copy number, we assessed the effect of exogenously added plasmid DNA in PCR reactions containing genomic DNA on PCR amplification of a DNA segment. This process can be formulated as follows:
Numbers of molecules in 3-week-old rosette leaves of wild-type Arabidopsis.
Genomic copy number/μl
2.47 × 105
2.40 × 105
4.12 × 105
4.39 × 105
0.16 × 105
0.22 × 105
0.47 × 105
0.33 × 105
1.48 × 1013
1.57 × 1013
2.40 × 1013
2.53 × 1013
0.05 × 1013
0.18 × 1013
0.36 × 1013
0.18 × 1013
5.99 × 107
6.54 × 107
5.83 × 107
5.76 × 107
2.60 × 108
2.84 × 108
2.54 × 108
2.51 × 108
3 RBC-L number/cell
2.63 × 10 8
0.15 × 10 8
5.70 × 10 -2
9.88 × 1013
10.01 × 1013
17.72 × 1013
19.78 × 1013
0.81 × 1013
0.20 × 1013
0.15 × 1013
0.04 × 1013
4.00 × 108
4.17 × 108
4.30 × 108
4.51 × 108
1.74 × 109
1.81 × 109
1.84 × 109
1.96 × 109
3 Chlorophyll number/cell
1.85 × 10 9
0.09 × 10 9
4.86 × 10 -2
We quantified the amount of RBC-L according to the intensity of CBB staining following SDS/PAGE analysis. However, it is also possible to use immunoblotting or enzyme-linked immunosorbent assay to quantify the number of molecules of a particular protein per genomic DNA copy number.
In this study, we demonstrate that establishing an appropriate normalization factor is a challenging yet vital aspect of comparing protein, transcript, or metabolite levels among samples. Our results establish a facile and accurate method for quantifying these molecules based on genomic DNA copy number and ploidy. Although we performed cytometric analysis to measure the ploidy, a simplified method without the cytometric analysis may be applicable when assessing the effect(s) of a short-term treatment (e.g., induction of stress over several hours). Furthermore, our method can provide information on the number of transcripts, proteins, and metabolites per cell, and it should be applicable for stoichiometry and mathematical modeling of cellular systems.
Arabidopsis thaliana, Wassilewskija (wild type) and abc4 (Wassilewskija background) were grown at 23°C under continuous light (32.0 μmol·m-2s-1) on plates containing 1/2 MS medium and 1.5% sucrose.
Flow cytometry was performed by a Ploidy Analyzer (Partec, Münster, Germany) . At least 5,000 nuclei isolated from rosette leaves of each 3-week-old Arabidopsis plant were used for each ploidy measurement. Three biological and two technical replicates were used for each sample analyzed.
Preparation of total nucleic acid from A. thaliana
Rosette leaves (100 mg) from each 3-week-old Arabidopsis plant were ground with a mortar and pestle in liquid nitrogen and homogenized in 5 volumes (v/w) of extraction buffer (100 mM MOPS-KOH, pH 7.0, 10 mM EDTA, 0.3 M NaCl, 1.0% SDS). Extraction of total nucleic acids (genomic DNA and total RNA) was performed by the addition of 5 volumes of phenol saturated with 1 M MOPS-KOH, pH 7.0, and 5 volumes of chloroform:isoamyl alcohol (24:1, v/v) followed by vigorous agitation and centrifugation for 10 min at 20,000 × g. The aqueous phase was collected and extracted two times with an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1, v/v/v). The nucleic acids were precipitated with ethanol and suspended in 50 μl nuclease-free water (see additional file 7).
Preparation of cDNA and genomic DNA
To prepare cDNAs, 4 μl total nucleic acid (A260 = 20.0) was digested with Turbo DNA-free DNase I (Ambion, Austin, TX, USA) according to the manufacturer's instructions. Absence of genomic DNA in DNase I treated samples was verified by PCR using primers T7F6-F-2 and T7F6-R-2 or MDC16-F-2 and MDC16-R-2 (see additional file 1). cDNA synthesis was performed using the PrimeScript RT reagent kit in the presence of oligo dT and random 6-mer primers according to the manufacturer's instructions (Takara Bio. Inc., Ohtsu, Japan). To prepare genomic DNA, 4 μl total nucleic acid (A260 = 20.0) was digested with RNase (Wako Pure Chemical Industries, Osaka, Japan) (see additional files 7 and 8).
Protein sample preparation
Rosette leaves (100 mg) from each 3-week-old plant were ground with a mortar and pestle in liquid nitrogen and transferred to a new 2-ml tube and homogenized in 5 volumes (v/w) of extraction buffer containing 15 mM Tris-HCl, pH 8.0, 50 mM NaCl, 10 mM EDTA, 1.0% SDS and 1.0% protease inhibitor cocktail (Sigma-Aldrich, Tokyo, Japan). The samples were incubated on ice for 10 min with vigorous vortexing every minute. The samples were centrifuged for 5 min at 10,000 × g, and the supernatants were used for further experiments. The total protein concentration of each supernatant was determined using a Coomassie Protein Assay kit (Pierce Biotechnology, Rockford, IL, USA). Chlorophyll concentrations were calculated according to Arnon .
All qPCR reactions were analyzed with an ABI PRISM 7300 sequence detection system (Applied Biosystems, Foster City, CA, USA), and data were analyzed using SDS 2.2.1 software (Applied Biosystems). The KAPA SYBR FAST qPCR kit (Kapa Biosystems, Boston, MA, USA) was used for qPCR amplification of purified genomic DNA or cDNA according to the manufacturer's instructions; the 20-μl reactions contained 1 μl of genomic DNA or cDNA template and 8 pmol of each set of gene-specific primers (see additional files 1). PCR reaction conditions were as follows: 95°C for 3 min, followed by 40 cycles of 95°C for 15 s and 60°C for 40 s. Transcript levels were quantified by the ΔΔCt method . The KAPA2G Robust HotStart kit (Kapa Biosystems) was used for qPCR amplification of genomic DNA in protein extract supernatants; the 20-μl reactions contained 4 μl of undiluted KAPA2G Buffer B, 4 μl of undiluted Enhancer 1, 1.6 μl of 2.5 mM dNTP, 8 pmol of each gene-specific primer (T7F6-3-F and T7F6-3-R, see additional file 1), 1 μl of 0.1% SYBR Green I (Takara), 0.4 μl Rox High (from the KAPA SYBR FAST qPCR kit), 2 U of KAPA2G Robust HotStart DNA polymerase, and 1 μl of protein extract. For quantification of genomic DNA copy number in the mutant plants relative to wild-type plants, 1 μl of protein extract of wild-type plants (diluted 1:10, 1:20, 1:40, 1:80 or 1:160) was added to the PCR reactions, and a standard curve was generated by qPCR (see additional file 5). One microliter of a 1:40 dilution of protein extract of the mutant plants was added to the PCR reactions, and qPCR was performed. For absolute quantification of genomic DNA copy number in protein extracts of wild-type plants, 1 μl of a 1:40 dilution of wild-type protein extract and 1 μl of cloned PCR products (between 2.88 × 102 and 2.88 × 109 molecules, or 0 molecules) was added to the PCR reactions; the 20-μl reactions contained 4 μl of undiluted KAPA2G Buffer B, 4 μl of undiluted Enhancer 1, 4.4 μl of 25 mM MgCl2, 1.6 μl of 2.5 mM dNTP, 8 pmol of each gene-specific primer (T7F6-3-F and T7F6-3-R, see additional file 1), 10 pmol of TaqMan Probe (see additional file 1), 0.4 μl Rox High (from the KAPA SYBR FAST qPCR kit), 2 U of KAPA2G Robust HotStart DNA polymerase, and 1 μl of protein extract, and PCR was performed. This dilution series was prepared with EASY Dilution (for real-time PCR) (Takara). PCR reaction conditions were as follows: 95°C for 3 min, followed by 40 cycles of 95°C for 15 s and 72°C for 40 s.
Preparation of cloned PCR products
PCR was performed using primers T7F6-3-F and T7F6-3-R (see additional file 1) with genomic DNA of wild-type Arabidopsis as template. The amplified DNA fragment was ligated into the TA cloning vector, pMD20 (Takara), and the sequence was confirmed. The plasmids containing the PCR products were digested with Xho I and purified. The mass of a nucleotide pair in DNA is 660 Da, and the plasmid containing the PCR product was 2,872 bp. The concentration of the linearized plasmid was determined, and the number of plasmid molecules was calculated.
Expression and purification of RBC-L
The full-length open reading frame of RBC-L was amplified by PCR using primers containing an Nde I site (RBCL-Nde, 5'-CCCCATATG TCACCACAAACAGAGACTAAAG-3'; Nde I site underlined) and a Xho I site (RBCL2-Xho, 5'-CCCCTCGAG CTCTTGGCCATCTAATTTATCGATG-3'; Xho I site underlined). The amplified DNA fragment was digested with Nde I and Xho I and ligated into the expression vector pET-24a(+) (Novagen, San Diego, CA, USA). RBC-L was expressed and purified as described . The molecular weight of RBC-L was 54,019. The concentration of the purified RBC-L was determined, and the number of RBC-L molecules was calculated.
List of abbreviations
light-harvesting chlorophyll a/b protein
quantitative reverse transcription PCR
- RBC or Rubisco:
ribulose 1,5-bisphosphate carboxylase/oxygenase
large subunit of Rubisco
small subunit of Rubisco
- ACT2 :
- PDF2 :
transposable element gene
- SAND :
SAND family protein
- GAPDH :
glyceraldehyde 3-phosphate dehydrogenase
- UBC :
- EF-1α :
elongation factor 1-α
- PPR :
pentatricopeptide repeat-containing protein
- YLS8 :
yellow-leaf-specific protein 8
- UBC9 :
ubiquitin-conjugation enzyme E2
We thank T. Shibata (Hiroshima University) for critical reading of the manuscript. The Plant culture facility was provided by the Center for Gene Science, Hiroshima University. This work was supported by the Japan Society of the Promotion of Science [Grant-in-Aid for Scientific Research (c) (No. 20570032 to HS)].
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