Application notes
Vol.1 Comparisons of Sensitivity of Whole Yeast Genomic DNA Chips
DNA Chip Research Inc.
Toray Industries Inc.
We present here examples of gene expression analysis in which 3D-Gene® Yeast Oligo chip 6k was used. This chip is an entire yeast genomic chip composed of a columnar structure. On this chip, probes are mounted for 5,888 genes registered in Saccharomyces Genome Database(SGD). Gene expression of the budding yeast (Saccharomyces cerevisiae) can be comprehensively analyzed.
First, a gpd1 deleted yeast strain in 5 mL YPAD medium was shaken overnight at 30℃ (the preculture). Then 150 mL of YPAD medium was inoculated so that the OD660 was 0.1, and it was shaken and cultured at 30℃ (the main culture). The cells were harvested when the cells growth corresponded to OD660=0.6. Total RNA of the resulting cells was adjusted using the method of Schmitt et al.1).Then aRNA synthesis and labeling were performed. For gpd1 gene spiking, mRNA was synthesized by in vitro transcription of cDNA. Then aRNA amplification and labeling were performed. Each adjusted aRNA was mixed in a fixed proportion, and comparative analysis of sensitivity was performed by competitive hybridization.
Fig. 1 shows portions of images after hybridization. The S/N ratio of our DNA chip was over 3 times that of the DNA chip from Company B at the same dilution step. Thus, our chip is anticipated to accurately detect genes considered to be below the detection limit of a conventional chip.
Fig. 2 shows the detection sensitivity of our chip. It can not only detect one molecule of gene per cell (dilution series 1/300K), but also less than one molecule. That is, our chip can detect very small changes occurring in some cells among the harvested yeast cells.
| 1:150K | |
|---|---|
| Company B Protocol:Company B |
361(S/N:3)
|
| 3D-Gene Protocol: 3D-Gene |
1467(S/N:10)
|
Fig.1 Comparison of sensitivity with a chip from Company B
Yeast is one of the organisms which have been studied the longest. There is a wealth of knowledge on various factors in yeast including signaling factors. There are maintained databases with these factors. In the future, functions need to be comprehensively elucidated for genes with ultra-low expression using probes with high specificity and DNA chips with high sensitivity. In addition, databases need to be enhanced. It can be anticipated that then yeast will be the "model organism" of eukaryotic cells which can be used in worldwide research of further wide-ranging fields.
Fig.2 Images of 3D-Gene® Yeast Oligo chip 6k sensitivity
References:
- 1) Schmitt, et al. (1990) Nucleic Acids Res, 18: 3091-3092.