Novel DNA microarray system for analysis of nascent mRNAs

Abstract

Transcriptional activation and repression are a key step in the regulation of all cellular activities. The development of comprehensive analysis methods such as DNA microarray has advanced our understanding of the correlation between the regulation of transcription and that of cellular mechanisms. However, DNA microarray analysis based on steady-state mRNA (total mRNA) does not always correspond to transcriptional activation or repression. To comprehend these transcriptional regulations, the detection of nascent RNAs is more informative. Although the nuclear run-on assay can detect nascent RNAs, it has not been fully applied to DNA microarray analysis. In this study, we have developed a highly efficient method for isolating bromouridine-labeled nascent RNAs that can be successfully applied to DNA microarray analysis. This method can linearly amplify small amounts of mRNAs with little bias. Furthermore, we have applied this method to DNA microarray analysis from mouse G2-arrested cells and have identified several genes that exhibit novel expression profiles. This method will provide important information in the field of transcriptome analysis of various cellular processes.

Figure 1

Overview of the isolation of nascent RNAs for microarray analysis.

Figure 2

Binding specificity of BrU-labeled cRNA by in vitro transcription. (A) eGFP and luciferase gene were transcribed in vitro by T7 RNA polymerase. Br-UTP was used as the substrate instead of UTP during the transcription of eGFP. (B) Mixture of 200 ng of BrU-labeled eGFP and non-labeled luciferase cRNA was immunoprecipitated with addition to blocking agents. The eluted cRNAs were converted into cDNA, and then the cDNA copy number was measured using real-time quantitative PCR. The blocking agents used are as follows: (i) no addition, (ii) uridine, (iii) E. coli 16S and 23S RNAs, and (iv) total RNA from mouse FM3A cells. ‘Ratio (eGFP/Luci.)’ represents the ratio of the eGFP cDNA copy number to the luciferase copy number. Error bars represent standard errors of the means (n = 3).

Figure 3

Binding specificity in immunoprecipitation with spike-in controls. (A) Overview of the experiment. (B) Br-eGFP and luciferase cRNA were prepared as spike-in controls. ‘Ratio (eGFP/Luci.)’ represents the ratio of the eGFP cDNA copy number to the luciferase copy number. Error bars represent standard errors of the means (n = 3).

Figure 4

(A) Eluted RNAs were converted to cDNA, and cDNA was amplified by TS-PCR. (B) Validation with quantitative RT–PCR of the DNA microarray data using each amplification method. Genes expressed at various levels in the non-amplification method, TS-PCR amplification method, and T7 in vitro transcription (IVT) amplification method were confirmed using quantitative RT–PCR. Quantitative RT–PCR was performed using the total RNA of these transcription factors. Gapd was used as the control. The same total RNA was used for quantitative RT–PCR and DNA microarray experiments. The ratio of the total target RNA levels was normalized by using those of the Tbp (Mus musculus TATA box binding protein) gene, and the fold change for each gene in the differentiated ES cells compared to the control ES cells was calculated. Error bars represent standard deviations of log (base 2) of ratio. (C) Amplified cDNA labeled with Cy3/Cy5 underwent self-self hybridization.

Figure 5

DNA microarray analyses of nascent RNAs. (A) FT210 cells were synchronized in the G₂ phase by incubation at 39 °C for 17 h. Cell cycle profiles of asynchronous cells and G₂-arrested cells were analyzed by propidium iodide staining and flow cytometry. (B) Scatter plots comparing the expression profiles of asynchronous cells with those of G₂-arrested cells in a nascent RNA profile (left panel) and a steady-state RNA profile (right panel). (C) The Venn diagrams show the overlap of genes whose expression levels changed more than twofold in the nascent RNA profile and the steady-state RNA profile.