LabAutomation 2006 - SLAS

TP71
Danhui Wang
Sigma-Aldrich Corporation
St. Louis, Missouri
dwang@sial.com
Where Laboratory Technologies Emerge and Merge
Co-Author(s)
Derek Douglas
Carol Kreader
Jennifer Van Dinther
Rafael Valdes-Camin
A High Throughput Approach for Rapidly Identifying Knockdown of Gene Expression
for Functional Profiling of Biological Processes
As genome sequencing projects for different organisms are completed, the identification and characterization of the gene function
becomes fundamental to understanding the complexity of these systems. This creates a need for comprehensive studies to fill the gap
between sequence and function. Many investigative approaches for identifying gene function have been introduced, but RNA interference
(RNAi) studies have gained the most prominence due to its potential to enable rapid genome-wide loss-of-function screens in mammalian
systems. RNAi experiments demand careful control of a wide range of variables, and require a high throughput approach for isolating and
quantifying mRNA levels to allow for optimization and validation of experimental parameters. Standard methods for isolating mRNA can be
laborious, time consuming, and not amenable to automation. An automated system has been developed for the isolation and subsequent
analysis of the mRNA. This system utilizes Sigma’s SpyLine Poly A+ Capture kit, a novel system for the rapid isolation of poly A+ mRNA
from cultured mammalian cells for direct use in quantitative reverse transcriptional PCR (RT-PCR) analysis. The automated method was
used to identify effective RNAi gene knockdown. Results indicate that this approach has both the sensitivity and reproducibility necessary
for measuring transcript levels following gene knockdown.
TP72
Jennifer Halcome
Eppendorf-5 Prime, Inc.
Boulder, Colorado
halcome.j@e5p.com
Co-Author(s)
JaNae Myers
George Halley
Lars E Peters
Eppendorf - 5 Prime, Inc.
Tatiana Oustich
University of Colorado Health Science Center
Automated Library Screening Using an In Vitro Plasmid Amplification System on the
Eppendorf Workstation
The streamlined automation of routine tasks such as library screening has become an important objective for researchers in many
fields. Eppendorf has developed a novel approach to rapid and efficient library screening. This approach pairs the epMotion 5075 MC,
a workstation that is completely integrated with a Mastercycler ep thermal cycler, and our new in vitro plasmid amplification system,
OriMaster. In this study, a neurotoxin cDNA library was prepared for sequencing using both OriMaster automated on the epMotion 5075
MC, and a traditional alkaline lysis plasmid purification automated on the epMotion 5075 Vac. The methods were compared with regards
to overall time, hands-on time, sequencing quality, and data reliability. Eppendorf OriMaster provided DNA ready for sequencing from
single bacterial colonies in about 18 hours less time than the overnight growth and purification. Once colonies were picked, this automated
amplification protocol required five minutes to set up—there were no filter plates or adapters to stack, and the workstation performed all
mixing and diluting. OriMaster created linear, double-stranded plasmid DNA copies that were easily screened on a gel to determine which
plasmids contained inserts before sequencing. OriMaster sequencing quality and read lengths were higher than the traditional plasmid
preparation. Over 100 different OriMaster clone sequences were aligned with, and found to be identical to, the corresponding sequences
of the traditional plasmid preparation. Finally, the clones were independently classified for each preparation method and these classification
schemes were found to be congruent.
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