LabAutomation 2006 - SLAS

TP83
Bruce Seligmann
HTG
Tucson, Arizona
bseligmann@htgenomics.com
Where Laboratory Technologies Emerge and Merge
Co-Author
Ralph Martell, HTG Tucson
qNPATM Gene Expression Cell and Tissue-based Assays for Target Validation,
Screening, EC50-Based Assessment and Optimization of Efficacy, Specificity,
Metabolism and Safety
The quantitative Nuclease Protection Assay (qNPATM) is a microplate-based multiplexed assay technology for measuring gene expression.
The reproducibility of qNPA, providing whole cell or tissue assay average CV’s ~10% (between biological samples and thus including cell
or animal treatment variability), with repeatable day-to-day with >85% certainty, enables precise determinations of gene levels between
differently treated samples in vitro and in vivo. qNPA also allows for accurate measurement of time course and dose response curves
from which precise EC50 values can be calculated. By utilizing a lysis only protocol without the need for extraction or gene amplification,
small samples can be tested, thousands per day in high throughput, Therefore, qNPA can be used to measure gene expression from any
sample, including fixed tissue, whole blood, and melanoma tissue which are examples of samples that may prove to be difficult for other
methods.
An example of EC50-based gene expression QSAR will be presented.
The qNPA gene expression assay has the advantage over protein assays that all metabolism pathways can be efficiently assessed in a
cost effective manner, yet delivers a high reproducibility. Both in vitro and in vivo predictive qNPA tox models will also be discussed.
In summary, qNPA can be applied to all stages of target validation, drug discovery, and used to assess drug efficacy, specificity,
metabolism and safety in the same dose response manner. It can also have the same precision as proteins simply by just changing the
genes monitored.
TP84
Kerstin Thurow
University Rostock
Rostock, Germany
kerstin.thurow@uni-rostock.de
Co-Author(s)
Daniel Haller
Norbert Stoll
celisca
Establishment of an Automated Procedure for Viral RNA Isolation From Cell-Free
Bovine Samples
During the recent years, detection of viruses, bacteria or other pathogens in a variety of different samples was put into focus of scientific
and ecological interest. By using a High Throughput Screening platform thousands of samples might be examined within a single run.
Serum samples of infected cattle are investigated on the presence of viral RNA on a robotic core facility asset in a 96-well format.
Therefore magnetic beads are automatically subjected to the serum sample and incubated for 5 minutes . The beads are dislodged
from the liquid phase and attracted to the wall of each cavity. The RNA-free supernatant is removed automatically (Biomek NX Span-8,
Beckman Coulter) and beads carrying target RNA are washed several times in wash buffer. Finally the viral RNA is eluted in 30 µl low salt
buffer and transferred into a clean RNAse-free target plate. Automated measurement of RNA concentrations is performed by appliance
of a dye emitting a fluorescence signal after complexing with RNA. The total concentration is obtained relatively to a standard curve on a
fluorescence reader.
First simulation results display high recovery rates of control RNA. However, additional test are imperative to evaluate the system and to
adjust the pipetting strategy to the needs of liquid parameters. As intended the automation of the assay has shown to be a time – effective
tool for future high throughput applications. Since the assay method is not limited to the selected viral RNA but applicable to virtually all RNA
and DNA species an extension of the method onto other samples e.g. blood, tissue or cells is possible and will be done in the near future.
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