Absolute quantification of the budding yeast transcriptome by means of competitive PCR between genomic and complementary DNAs

Abstract

Background: An ideal format to describe transcriptome would be its composition measured on the scale of absolute numbers of individual mRNAs per cell. It would help not only to precisely grasp the structure of the transcriptome but also to accelerate data exchange and integration.

Results: We conceived an idea of competitive PCR between genomic DNA and cDNA. Since the former contains every gene exactly at the same copy number, it can serve as an ideal normalization standard for the latter to obtain stoichiometric composition data of the transcriptome. This data can then be easily converted to absolute quantification data provided with an appropriate calibration. To implement this idea, we improved adaptor-tagged competitive PCR, originally developed for relative quantification of the 3'-end restriction fragment of each cDNA, such that it can be applied to any restriction fragment. We demonstrated that this "generalized" adaptor-tagged competitive PCR (GATC-PCR) can be performed between genomic DNA and cDNA to accurately measure absolute expression level of each mRNA in the budding yeast Saccharomyces cerevisiae. Furthermore, we constructed a large-scale GATC-PCR system to measure absolute expression levels of 5,038 genes to show that the yeast contains more than 30,000 copies of mRNA molecules per cell.

Conclusion: We developed a GATC-PCR method to accurately measure absolute expression levels of mRNAs by means of competitive amplification of genomic and cDNA copies of each gene. A large-scale application of GATC-PCR to the budding yeast transcriptome revealed that it is twice or more as large as previously estimated. This method is flexibly applicable to both targeted and genome-wide analyses of absolute expression levels of mRNAs.

Figure 1

Generalized Adaptor-Tagged Competitive PCR (GATC-PCR). (A) Gene-specific primer (GSP)-dependent amplification from Y-shaped adaptor-tagged template. (B) An example of GATC-PCR. Genomic DNA and cDNA digested with Mbo I were ligated with adaptor A/C and B/C (Table 2), respectively, and used for GATC-PCR. The products of four assays (blue, green, red, and black) and a size standard (orange) were separated on ABI 3730 Genetic Analyzer. The fast- and slow-migrating peaks of each pair correspond to the signals from genomic DNA and cDNA, respectively. (C) Linearity of GATC-PCR from genomic DNA templates. Genomic DNAs extracted from the wild and gcn4Δ cells were combined at appropriate ratios to prepare a series of genomic DNAs containing 0, 0.25, 0.5, 0.75, and 1 copy of GCN4 per haploid on average, digested with Mbo I, and ligated to the adaptors A/C and B/C (Table 2). Various combinations of the A/C- and B/C-tagged templates were mixed in a 1:1 ratio, while keeping the total amount equivalent to 3,000 haploid cells, and subjected to GATC-PCR using a GCN4-specific primer. (D) Linearity of GATC-PCR from cDNA templates. An experiment similar to the one shown in (C) was conducted using cDNAs, instead of genomic DNA, prepared from the wild and gcn4Δ cells.

Figure 2

Calibration of GATC-PCR between genomic DNA and cDNA. (A) Competitive amplification of GCN4 between genomic DNA and cDNA. (B) Standard RNAs used for competitive PCR determination of mRNA copy number. (C) Comparison of absolute amounts of eight mRNAs determined by real-time PCR and GATC-PCR. For real-time PCR, we used each GSP for the first strand cDNA synthesis. The GATC-PCR data were calibrated by the competitive PCR quantification of GCN4 mRNA using the standard RNA set (Figure 2B, Table 3).

Figure 3

Absolute quantification of the budding yeast transcriptome by large-scale GATC-PCR. (A) Reproducibility of absolute quantification of the budding yeast transcriptome by GATC-PCR. Genome-wide GATC-PCR quantification was performed twice using the same total RNA sample labeled as #2 in Table 1. (B) ''Virtual R₀t'' curve based on the merged expression data (Table 4). (C) Comparison of absolute mRNA levels between cells grown in YPD and SD media. Note that the plot includes 3,351 genes detectably expressed under both conditions but not those with undetectable levels of expression in either condition. (D) Comparison of absolute mRNA levels of genes with GO slim term ''ribosome'' between cells grown in YPD and SD media. (E) Distribution of transcript abundances in cells grown in YPD and SD media. The plot includes 3,351 genes detectably expressed in both conditions.