Miniaturization of a functional transcription assay in yeast (human progesterone receptor) in the 384- and 1536-well plate format.
Miniaturization of high throughput screening assays to high-density microplate formats (384 or 1536 wells) is currently the focus of research activity in modern drug discovery facilities. In this article, we describe the adaptation of a fluorescence-based functional transcription assay in yeast for assessing modulators of human progesterone receptor to the 384- and 1536-well microplate format, comparing the experimental results to those obtained in the well-established 96-well format. The experiences gained from the optimization of the liquid-handling procedures and the miniaturization of an enzyme assay (beta-galactosidase) were implemented. Thus optimized pipetting protocols were developed to perform a reporter gene assay in yeast in microplate formats of higher density. In the functional transcription assay in yeast, the reporter gene expression showed the expected dependence on the ligand's dose and affinity in principle in all three microplate formats. For the first time, this assay system has been established in the 1536-well microplate format using CyBi-Well 96/384/1536 as the liquid-handling unit. The comparison of the signal:background ratios showed a lower sensitivity of the assay in the microplate formats of higher density. This study is an example of a successful miniaturization of a yeast cell-based assay to high-density plate formats on the basis of a careful adaptation procedure and optimized liquid-handling conditions.
Berg
M
M
Hans-Knöll-Institut für Naturstoff-Forschung e.V., Jena, Germany.
Undisz
K
K
Thiericke
R
R
Moore
T
T
Posten
C
C
D003001:Cloning, Molecular; D017930:Genes, Reporter; D006801:Humans; D008904:Miniaturization / Q000295:instrumentation / Q000379:methods; D011980:Receptors, Progesterone / Q000032:analysis* / Q000235:genetics; D011994:Recombinant Proteins / Q000032:analysis; D012441:Saccharomyces cerevisiae / Q000235:genetics; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000295:instrumentation / Q000379:methods; D014158:Transcription, Genetic; D001616:beta-Galactosidase / Q000032:analysis / Q000235:genetics
D011980:Receptors, Progesterone; D011994:Recombinant Proteins; D001616:beta-Galactosidase