After yeast cells were grown for 6 h (exponentially growing cells) at 30℃, 5 mg of YCl3・6H2O (Wako Pure Chemical Industries, Osaka, Japan) or 3 mg, 20 mg of Y2O3 nanoparticle (CIK NanoTek Corporation, Tokyo, Japan) were exposed to yeast cells, and these yeast cells were further incubated for 2h.
Growth protocol
These yeast cells were grown in YPD medium (1.0% yeast extract, 2.0% peptone ; Becton, Dickinson and Co., NJ, USA, and 2.0% glucose, if necessary 1.5% agar; Wako Pure Chemical Industries, Ltd, Osaka, Japan) at 30°C for 8 h.
Extracted molecule
total RNA
Extraction protocol
The yeast cells after stressed were collected by centrifugation of 15,000 rpm for 1.0 min at 4°C. RNA extraction conformed according to partially modified manufacture protocol of Fast RNA ® Pro Red Kit (MP Biomedicals, CA, USA). RNA was quantified using a NanoDrop-1000 spectrophotometer and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
Label
Cy3
Label protocol
The cDNA was synthesized by Quick Amp Labeling Kit (Agilent) using 0.5 μg total RNA as a template. Cyanine-3 (Cy3) labeled cRNA was prepared from cDNA using the T7 RNA polymerase. Cy3-labeled cRNA was purified by RNeasy Mini Kit (QIAGEN, Hilden, Germany), and checked with the NanoDrop ND-1000 Spectrophotometer.
Hybridization protocol
Cy3-labelled cRNA was fragmented at 60°C for 30 min in a reaction volume of 25 μl containing 1x Blocking Agent and 1x Fragmentation Buffer. On completion of the fragmentation reaction, 2x Hybridization Buffer HI-RPM solution was added to the fragmentation mixture containing 0.6 μg of Cy3-labelled cRNA and hybridized for 17 h, at 65C and 10 rpm. After hybridization, microarrays were washed according to Quick Amp Labeling Kit Protocol (Agilent).
Scan protocol
The hybridized microarray slides were washed according to the manufacturer’s instructions and were scanned with an Agilent DNA Microarray Scanner (#G2565CA, Agilent Technologies, CA, USA) at a resolution of 5 μm.
Data processing
These data were normalized by the quantile methods using Excel.
