Paterson Institute for Cancer Research SCIENTIFIC REPORT 2005

54
RESEARCH SERVICES
mised to detect stress activation in mouse embryos
including phosphorylated p38 kinase or hypoxia
specific marker HIF1α.
The unit, together with the Clinical and experimental
pharmacology group, has also been involved in
the optimisation of immunohistochemical (IHC)
methodologies for cleaved caspase 3, PARP and
cytokeratin 18 for use in pre-clinical pharmacodynamic
IHC assays. The assays focus on detecting
and quantifying levels of apoptosis in cells and tissue
which will eventually be used in the clinical trial
of agents which modulate/inhibit apoptotic pathways.
A new sucrose/gelatin method for the preparation
and freezing of embryoid bodies and cell preparations
has been implemented. The method both cryoprotects
and immobilises specimens allowing for
much improved morphology and numbers.
Single 0.6mm core of a gastric tumour from a tissue microarray
The unit recently purchased a tissue microarray
(TMA) platform, allowing for the generation of
multiple specimen slides. The platform is an essential
tool in the shift towards translational research,
conserving valuable material whilst allowing standardisation
of methodology. TMAs of both gastric
tumours and normal breast tissue have already been
constructed and this service will continue to be
expanded with the construction of TMAs from
mouse models.
Advances in the isolation and extraction of quality
RNA based on immunophenotype have been
achieved. In parallel with this, work on the possible
recovery of material from archival formalin-fixed
paraffin-embedded specimens will be assessed over
the coming months. This technique has the possibility
of enabling the gene expression analysis of
huge numbers of previously inaccessible samples.
Another area of interest to the unit is quantum dot
technology. Methodologies for direct antibody
conjugation and the use of Q dot bioconjugates will
be assessed and optimised, allowing for multilabelling
and accurate signal quantification of tissue
sections.
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Molecular Biology Core Facility.
Head: Stuart Pepper.
http://www.paterson.man.ac.uk/facilities/mbcf.jsp
The molecular biology core facility provides access
for Paterson staff to a range of state-of-the-art
technologies for analysis of DNA, RNA and protein.
Routine plasmid extraction and sequencing is
offered as a complete service where facility staff
carry out all the processing, whereas quantitative
PCR is offered as a technology platform for which
we offer technical support and advice. Over the last
year our capability to support qPCR has improved
significantly; we now have a 384-well pipetting system
available to users which allows larger experiments
to be tackled in a time effective manner.
Purchase of the Exiqon probe library has also
streamlined the design phase of qPCR projects,
meaning that users are able to start generating useful
results very quickly. As 2005 draws to a close we
are starting to offer an assay validation service in
which we will design and validate assays to specific
genes for users so that the first experiment a user
performs will be to generate the results they want.
Last year the facility expanded into the area of protein
analysis by mass spectrometry. Developments
in this area have benefited immeasurably from the
support and expertise of the Stem Cell Leukaemia
and Proteomics Lab, University of Manchester.
The facility operates two machines that are routinely
delivering excellent results for our users: a Q Star
which offers excellent mass accuracy and resolution,
and is well suited to ‘routine’ identification of
sequencing of peptides, and a Q Trap which is a
unique hybrid instrument that is amenable to studies
on post-translational modifications such as
phosphorylation. In combination with 3 LC
Packings Ultimate Nano LC systems, two of which
are able to perform 2-dimensional chromatography,
these mass specs offer a very flexible system for the
comprehensive analysis of protein mixtures. Last
year these machines were used in a number of
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