An improved single-cell cDNA amplification method for efficient high-density oligonucleotide microarray analysis

A systems-level understanding of a small but essential population of cells in development or adulthood (e.g. somatic stem cells) requires accurate quantitative monitoring of genome-wide gene expression, ideally from single cells. We report here a strategy to globally amplify mRNAs from single cells for highly quantitative high-density oligonucleotide microarray analysis that combines a small number of directional PCR cycles with subsequent linear amplification. Using this strategy, both the representation of gene expression profiles and reproducibility between individual experiments are unambiguously improved from the original method, along with high coverage and accuracy. The immediate application of this method to single cells in the undifferentiated inner cell masses of mouse blastocysts at embryonic day (E) 3.5 revealed the presence of two populations of cells, one with primitive endoderm (PE) expression and the other with pluripotent epiblast-like gene expression. The genes expressed differentially between these two populations were well preserved in morphologically differentiated PE and epiblast in the embryos one day later (E4.5), demonstrating that the method successfully detects subtle but essential differences in gene expression at the single-cell level among seemingly homogeneous cell populations. This study provides a strategy to analyze biophysical events in medicine as well as in neural, stem cell and developmental biology, where small numbers of distinctive or diseased cells play critical roles

Quantitative expression profile of distinct functional regions in the adult mouse brain.

The adult mammalian brain is composed of distinct regions with specialized roles including regulation of circadian clocks, feeding, sleep/awake, and seasonal rhythms. To find quantitative differences of expression among such various brain regions, we conducted the BrainStars (B*) project, in which we profiled the genome-wide expression of ∼50 small brain regions, including sensory centers, and centers for motion, time, memory, fear, and feeding. To avoid confounds from temporal differences in gene expression, we sampled each region every 4 hours for 24 hours, and pooled the samples for DNA-microarray assays. Therefore, we focused on spatial differences in gene expression. We used informatics to identify candidate genes with expression changes showing high or low expression in specific regions. We also identified candidate genes with stable expression across brain regions that can be used as new internal control genes, and ligand-receptor interactions of neurohormones and neurotransmitters. Through these analyses, we found 8,159 multi-state genes, 2,212 regional marker gene candidates for 44 small brain regions, 915 internal control gene candidates, and 23,864 inferred ligand-receptor interactions. We also found that these sets include well-known genes as well as novel candidate genes that might be related to specific functions in brain regions. We used our findings to develop an integrated database (http://brainstars.org/) for exploring genome-wide expression in the adult mouse brain, and have made this database openly accessible. These new resources will help accelerate the functional analysis of the mammalian brain and the elucidation of its regulatory network systems

Acute Induction of Eya3 by Late-Night Light Stimulation Triggers TSHβ Expression in Photoperiodism

SummaryLiving organisms detect seasonal changes in day length (photoperiod) [1–3] and alter their physiological functions accordingly to fit seasonal environmental changes. TSHβ, induced in the pars tuberalis (PT), plays a key role in the pathway that regulates vertebrate photoperiodism [4, 5]. However, the upstream inducers of TSHβ expression remain unknown. Here we performed genome-wide expression analysis of the PT under chronic short-day and long-day conditions in melatonin-proficient CBA/N mice, in which the photoperiodic TSHβ expression response is preserved [6]. This analysis identified “short-day” and “long-day” genes, including TSHβ, and further predicted the acute induction of long-day genes by late-night light stimulation. We verified this by advancing and extending the light period by 8 hr, which induced TSHβ expression within one day. In the following genome-wide expression analysis under this acute long-day condition, we searched for candidate upstream genes by looking for expression that preceded TSHβ's, and we identified the Eya3 gene. We demonstrated that Eya3 and its partner Six1 synergistically activate TSHβ expression and that this activation is further enhanced by Tef and Hlf. These results elucidate the comprehensive transcriptional photoperiodic response in the PT, revealing the complex regulation of TSHβ expression and unexpectedly rapid response to light changes in the mammalian photoperiodic system

Schematic diagram of the key features of the global cDNA amplification method

<p><b>Copyright information:</b></p><p>Taken from "An improved single-cell cDNA amplification method for efficient high-density oligonucleotide microarray analysis"</p><p>Nucleic Acids Research 2006;34(5):e42-e42.</p><p>Published online 17 Mar 2006</p><p>PMCID:PMC1409679.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> () Evaluation system to verify representation of amplified cDNA from diluted ES cellular RNA by Q-PCR and/or microarray. () Gene representation distorted during the global PCR. Diluted ES cellular RNA (10 pg) was amplified as described elsewhere (), and the replicates of amplification were sequentially sampled at 16, 20, 24, 28, 32, 36, 40 and 44 cycles. The expression levels of , , , , and were measured by Q-PCR, normalized by that of , and represented with brown, cyan, yellow, blue, pink and green lines, respectively. The averages of four independent experiments are plotted. () Schematic diagram of cDNA amplification. The mRNA and cDNA are colored pink and orange, respectively. The V1, V3 and T7 promoter sequences are represented by blue, red and green boxes, respectively. The bars above the letters represent the complementary sequences

Direct application of the newly developed method to single ICM cells from mouse E3

<p><b>Copyright information:</b></p><p>Taken from "An improved single-cell cDNA amplification method for efficient high-density oligonucleotide microarray analysis"</p><p>Nucleic Acids Research 2006;34(5):e42-e42.</p><p>Published online 17 Mar 2006</p><p>PMCID:PMC1409679.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p>5 blastocyst reveals the presence of two distinct cell populations. () Hierarchical clustering of single ICM cells. () Heat map representation of differentially expressed genes (top 100). The expression levels are color-coded from red (high) to blue (low). The expression levels are normalized in the lows. () The correlation of gene expression is preserved between E3.5 and E4.5. The copy numbers of expressed genes were estimated with Q-PCR. Orange, pink and green bars represent high, middle and low/non-detectable expression of , respectively. -values of the Chi-square test for independence from expression are indicated. (D and F) Blastocysts at E3.5 () and E4.5 (). The typical embryos used for single-cell experiments are shown. (E and G) Expression levels of key genes related to PE and epiblast at E3.5 () and E4.5 (). All of the single-cell samples of ICMs are shown. The representation code is the same as in (C)

A functional genomics strategy reveals clockwork orange as a transcriptional regulator in the Drosophila circadian clock

The Drosophila circadian clock consists of integrated autoregulatory feedback loops, making the clock difficult to elucidate without comprehensively identifying the network components in vivo. Previous studies have adopted genome-wide screening for clock-controlled genes using high-density oligonucleotide arrays that identified hundreds of clock-controlled genes. In an attempt to identify the core clock genes among these candidates, we applied genome-wide functional screening using an RNA interference (RNAi) system in vivo. Here we report the identification of novel clock gene candidates including clockwork orange (cwo), a transcriptional repressor belonging to the basic helix–loop–helix ORANGE family. cwo is rhythmically expressed and directly regulated by CLK–CYC through canonical E-box sequences. A genome-wide search for its target genes using the Drosophila genome tiling array revealed that cwo forms its own negative feedback loop and directly suppresses the expression of other clock genes through the E-box sequence. Furthermore, this negative transcriptional feedback loop contributes to sustaining a high-amplitude circadian oscillation in vivo. Based on these results, we propose that the competition between cyclic CLK–CYC activity and the adjustable threshold imposed by CWO keeps E-box-mediated transcription within the controllable range of its activity, thereby rendering a Drosophila circadian clock capable of generating high-amplitude oscillation