omation mbers - Society for Laboratory Automation and Screening

WP009
Marcy Engelstein
Millipore Corporation
R & D
17 Cherry Hill Drive
Danvers, Massachusetts 01923
marcy_engelstein@millipore.com
Streamlining Automation of In-Gel Digestion and MALDI Target Spotting
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Co-Author(s)
Libby Kellard and Anja Dedeo, Millipore Corporation
Mikkel Nissum, Tecan
With the completion of the Human Genome Project, there is now significant emphasis on identifying proteins and
protein function. As a consequence, the number of protein samples being processed has increased dramatically.
Proteins are typically separated by MDLC or 2D gel electrophoresis. Those of interest are digested, concentrated/
desalted, and analyzed using mass spectrometry. This procedure is time consuming and labor intensive. We
describe here two methods to improve the In-Gel digestion process – a one-plate or two-plate protocol that
differ primarily in their throughput and automation capabilities of the elution step. The one plate protocol uses the
Montage ® In-Gel Digest ZP Kit (Millipore) on the Tecan Genesis ® . This procedure allows for automated processing
of gel pieces in a ZipPlate micro-SPE plate up to the C18 elution step. Peptides are eluted and transferred directly
to the MALDI target off-line. A fully automated process is available with this protocol using vacuum elution with
subsequent spotting onto the MALDI target. The two-plate protocol utilizes a ZipPlate ® micro-SPE plate (Millipore)
in tandem with a TecPro96 plate (Tecan) on the Tecan Genesis. This procedure allows for full automation of the
entire process, including direct transfer of purified peptides from the ZipPlate plate onto a MALDI target plate for
immediate analysis.
WP010
Xingwang Fang
Ambion, Inc.
Research and Development
2130 Woodward Street
Austin, Texas 78744
xfang@ambion.com
High Throughput Sample Preparation for RNAi Studies and Expression Profiling
Co-Author(s)
Roy C. Willis, Quoc Hoang,
Michael Siano, Weiwei Xu
The demand for robust high throughput RNA isolation and amplification increases very rapidly since RNAi
and microarray technologies gains wide applications. We present: 1) high throughput siRNA synthesis by in
vitro transcription; 2) a comparison of various high throughput RNA isolation; and 3) a fully automated mRNA
amplification method for microarray analysis. In vitro transcription can generates both short (21bp) and long
(>300bp) double-stranded RNAs with same function as chemical synthesized RNAs, but in much shorter time. The
protocol is automatable in 96- or 384-well format, enabling quick screening for the best sequence to targeting a
specific gene. Different high throughput RNA isolation methods will be compared, focusing on consistency and
quality of isolated RNA and simplicity and robustness of the protocol. We’ll also discuss the streamline of RNA
isolation with RNA quantification by qRT-PCR and amplification for microarray analysis. In general, microspheric
bead-based approach results in more consistent RNA recovery than glass fiber filter based RNA method, and
RNA can be eluted in a smaller volume. This is because beads can be fully resuspended in solution to enable
more thorough mixing, washing, and elution, while the glass fiber matrix is fixed in a filter plate. In addition, we will
present a high throughput sample preparation for microarray analysis. The method is based on the Van Gelder and
Eberwine procedure and seamlessly integrates mRNA amplification, labeling and purification in a 96-well format.
The results from beta testing of this technology will be presented.