Improved toxicogenomic screening for drug-induced phospholipidosis using a multiplexed quantitative gene expression ArrayPlate assay.
We previously showed that a toxicogenomics analysis of drug-induced phospholipidosis enabled the identification of 12 specific gene markers and the establishment of an in vitro real-time PCR screening assay for the assessment of the phospholipidosis-inducing potential of compounds. The purpose of this study was to transfer our PCR-based assay into a 96-well microplate-based multiple mRNAs measuring assay (ArrayPlate assay) in order to increase throughput. Specifically, we determined the expression of the 12 marker genes using real-time PCR and ArrayPlate in human hepatoma HepG2 cells that were treated for 24h with each of amiodarone and 80 proprietary compounds. The following three performance criteria were satisfied in the ArrayPlate analysis: 1. Sensitivity-the expression of mRNA for all target genes was detected at quantifiable levels. 2. Repeatability-signal intensities and fold change values of each marker gene were highly repeatable. 3. Correlation-fold change values and their average values, which were used as indices of phospholipidosis induction potential, showed apparent correlation between the ArrayPlate and real-time PCR assays. Thus, the in vitro screening assay for compound-induced phospholipidosis should be transferable from a PCR-based assay to the higher-throughput ArrayPlate-based method.
Sawada
Hiroshi
H
Takeda Pharmaceutical Company Limited, Discovery Research Center, Pharmaceutical Research Division, 2-17-85 Juso-Honmachi Yodogawa-ku, Osaka 532-8686, Japan. Sawada_Hiroshi@takeda.co.jp
Taniguchi
Keiko
K
Takami
Kenji
K
D045744:Cell Line, Tumor; D015870:Gene Expression; D006801:Humans; D008064:Lipidoses / Q000139:chemically induced* / Q000235:genetics*; D046529:Microscopy, Electron, Transmission; D009152:Mutagenicity Tests; D020411:Oligonucleotide Array Sequence Analysis; D010743:Phospholipids; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction
D010743:Phospholipids; D012333:RNA, Messenger