Genomics Core Facilityand

Advanced Imaging Facilities

NewsletterThe buzz in your facilities

August 2011

The Genomics Core Facility and the Advanced Imaging Facilities

are dedicated to provide researchers with state-of-the art

equipment and technical/scientific support so they can perform

world-class studies. Work can be handed over to a member of

staff for completion or can be performed by the researcher after

training. For that reason, the facilities are also actively involved in

the training of students and users. Several workshops and

seminars are also organised during the year.

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Dear Reader,

The aim of this newsletter is to keep you informed of some of the

most recent and fizzy news which, we think, might be of interest

to you.

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•Focus on long term live cell imaging……………….……….…4-7

•Cheap MicroRNA profiling of 30 samples on Agilent microarrays……………………………………………………………..….8-9

•Real-Time Quantitative PCR for protein quantification ………………………………………………………………………………..10-11

•Get on Board of the new OpenArray, a new platform for large scale qPCR, genotyping and digital PCRs………...12-13

•Recent Studies and Publications…………..…………………14-15

•Express News………………………………………………………….16-17

•List of Equipment……………………………………………………18-19

•Staff Members…………………………………………………………….20

Contents

Contact DetailsGenomics Core Facility,University of Leicester, Adrian Building,Phone: +44 (0) 116 252 3375/3365Dr Nicolas Sylvius or Reshma VaghelaFor more details, visit website

Advanced Imaging FacilitiesCentre for Core Biotechnology Services, University of Leicester, Phone: +44 (0) 116 229 7085/252 2263 Dr Kees StraatmanFor more details, visit website

There have been remarkable advances in light microscopy and in fluorescence microscopy in particularly in the last decades. Only ten years ago you would need a special confocal laser scanning microscope for live cell imaging but advances in fluorochromedevelopment, detectors and computer speed and storage means that 24 hours live cell imaging experiments are now regularly performed on the confocal laser scanning microscopesin the AIF. Besides that we have also two, and soon three, wide field

microscopes specifically equipped for live cell imaging. All these microscopes have a temperature controlled culture chamber and we can flow 5% CO2 into this chamber. Depending on the system we can image cells on slides, dishes and multiwell plates.

In this article, we would like to introduce you to the various technical options you have in the facilities if you need to carry out imaging on live cells.

Focus on long term live cell imaging

The Leica TCS SP5 confocal laser scanning microscope.

The Olympus CellR system in the Adrian Building has an MT20 illumination system which contains a fast filter wheel (minimal switch 58 ms) and a fast attenuator with 14 grades of illumination intensity. The MT20 offers stabilised intensity for quantitative measurements. Filters are available for DAPI, FITC, YFP, GFP, CFP, TxRed, mRFPand Cy5. This system can also be used as a screening station when using the ScanR software. This can be done on both fixed and live cells, in dishes, slides or multiwell plates. To make the screening of slides easier the workshop has made stage inserts that can hold 4 slides.

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Nikon microscope 4 in the HWB is equipped with a LED light source for the wavelengths 400nm, 490nm and 565nm. Furthermore, we just ordered a LED light source for the Nikon TIRF microscope, housed in the MSB, what will allow us to do non-TIRF live cell imaging and imaging of fixed material on this system as well. We also will have an additional 635 nm LED for Cy5/Alexa633 imaging. Advantage of LEDs are fast switch-on switch –off, no warming up, very stable light source, long life span and low heat output. For signal detection both these microscopes are equipped with EM-CCD cameras for the detection of even the weakest signals. Both the Olympus CellR and the Nikon TIRF microscope have also a hardware autofocus system to keep your samples in focus during long term experiments.

For researchers who don’t use fluorescence but work with bioluminescence the AIF has the Olympus LV200 bioluminescence imaging station. This system is specifically developed for bioluminescence imaging on single cells or tissue and has sub-cellular resolution. A built-in system for temperature control, humidity, and gas flow helps to keep cultured cells or tissue slices healthy for long term (days) experiments. The system can handle small samples in 35 mm dishes.

The Olympus LV200 bioluminescence imaging system

In another development, a request has been made to get a Home Office licence for the Multi Photon Laser Scanning Microscope room in the MSB. This hopefully will be organized during the next visit by the Home Office Inspector. This system is equipped with a 5x overview objective and a 20x dipping objective to image in liquid environments. The advantage of a Multi photon system over a confocal laser scanning microscope is that it can image deeper into tissue. Where a normal confocalmicroscope can penetrate around 50 µm into tissue the MP system can penetrate to a depth of 1 mm into tissue. This system can also be used on fixed samples.

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Alternatives for fluorescent proteins in live cell imagingMost people use GFP or other fluorescent proteins (BFP, CFP, YFP, RFP) to tag their protein of interest for live cell imaging. In many cases this works fine. However, occasionally adding a fluorescent protein on the N- or C- terminal causes disruption of the function or localization of the tagged protein. As alternatives Covalys introduced the ACP-tag/MCP-tag for cell surface labelling and a SNAP-tag system to study proteins in live cells, commercialized by NEB. Another alternative is the FlAsH and ReAsHlabelling systems from Invitrogen . Both the ACP-tag and MCP-tag are 77aa (GFP = 238 aa) and are based on the Acyl Carrier Protein (ACP) from E. coli. MCP-tag contains two mutations compared with ACP to make it more substrate specific. Both tags can be detected using one of several CoA-based labelling substrates dependent on the

excitation you require. The SNAP-tag is a highly engineered version of the human form of O6- alkylguanine-DNA-alkyltransferase (AGT) of around 150 aa. NEB has released a whole range of para-substituted benzyl guanines bound to different fluorochromes for detection of the SNAP-tag.

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The Nikon TIRF microscope

Invitrogen's FiAsH and ReAsH uses a 6 aatag (Cys-Cys-Pro-Gly-Cys-Cys). FlAsH is derived from fluorescein and ReAsHfrom resorufin. They are virtually non-fluorescent when bound to ethane dithiol (EDT). When FlAsH-EDT or ReAsH-EDT bind to tetracysteine (TC) sequences, EDT is displaced and the tags become highly fluorescent in green or red, respectively. Besides the smaller tag these systems make it also easier to adapt your protocol when combined with fluorescent proteins or antibodies. By changing the substrate you change the colour used to detect your protein without changing the tag on your protein.

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Biogenesis and Mechanism of Action of MicroRNAs ,Yong Sun Lee and Anindya Dutta, Annual review of pathology, 2009

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MicroRNAs at a glanceMicroRNAs are endogenously expressed small non coding RNAs that target mRNAs and control post-transcriptional gene expression. They have emerged as critical players in multiple biological processes. A growing number of studies show that deregulation of microRNA expression and activity is implicated in numerous diseases which include infectious diseases, cancers, cardiovascular and neurological disorders.

Who needs MicroRNA transcriptome profiling?

The Genomics Core Facility managed to negotiate a VERY interesting deal with an external company to profile 30 human, mouse or rat samples on an Agilent microarray platform at a very attractive cost.

This service includes the quality control of the samples, the microRNA profiling and the bioinformatics analysis for ready-to-publish data. We can combine samples in batch of eight.

If you are interested, don’t hesitate to contact the Genomics Core Facility for more details.

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Promotion for microRNA profiling

Can Western Blots get shot by

TaqMan ?

Introducing the new TaqMan Protein

assays for protein quantification using

real-time qPCR

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Real-Time quantitative PCR for proteinLife Technologies (Applied Biosystems) has developed a new TaqMan® Protein Assay to quantify proteins by real-time quantitative PCR. You can make your own TaqMan® Protein Assay from a biotinylatedantibody for your protein of interest or you can buy pre-designed assays from Life Technologies.

This technology is still in its infancy but sounds very promising to complement or even replace laborious and antibody-consuming western blots and immunostaining. See Applied Biosystems for more details on this technique.

Try it for freeWe are keen to implement this new technique in the Genomics Facility.

If you are interested to give it a try, we can get free samples of the new TaqMan® Protein Assays. You will have to provide an aliquot of your samples and antibody. We can also arrange a field application scientist from Life Technologies to come in to help set up the protocol and we will install the required software on our machines for analysis of your data.

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OpenArray® Real-Time PCR System

The OpenArray system (Applied Biosystems) is a new PCR platform for real-time PCR, genotyping and digital PCR. It utilizes a microscope slide–sized plate with 3,072 through-holes. Each through-hole is 300 μm in diameter with a depth of 300 μm. The plates are arranged in 48 subarrays of 64 through-holes.

This platform has been developed for large scale genotyping and large scale qPCR -based gene quantification projects (Both TaqMan or SYBR®) which could not be achieved using regular qPCR instruments. It also allows you to perform digital PCR, a new technique for the absolute quantification of nucleic acids, based on PCR amplification of single target molecules. Therefore, digital PCR is the ideal technique for detecting/quantifying rare mutations, viral load, low-fold difference copy number variations, allelic imbalance detection, methylation status, next-gen sequencing library quantification and single-cell analysis. See ABI website for more details and our website to download the presentation ABI gave at the Facility last May.

If you need to carry out a large scale genotyping project or a large scale real-

time quantitative PCR-based project, Applied Biosystems is happy to lend the

Genomics Core Facility an OpenArrayplatform for the time of your project.

Various formats are available which will allow you to process from 120 samples (224 assays) to 480 samples (18 assays) or more. Contact Nicolas Sylvius or Reshma Vaghela.

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Briefly…

Recent studies and publications from users of the facilities

Dense genotyping of candidate gene Loci identifies variants associated with high-density lipoprotein cholesterol.Edmondson AC et al, Circ CardiovascGenet. 2011;4(2):145-55.High plasma levels of high-density lipoprotein cholesterol (HDL-cholesterol) are associated with decreased risk of atherosclerotic cardiovascular disorders. To have a better SNP coverage of HDL cholesterol candidate genes and to include low-frequency and rare nonsynonymousvariants, Edmondson and colleagues, and the department of cardiovascular sciences, used the Illumina humanCVDbeadchips, a dense genotyping microarray. This allowed them to genotype about 48,000 markers specifically focussed on genes associated with cardiovascular disorders in more than 7800 individuals. Not only did they confirm several of the loci identified in previous genome-wide association studies, but they also found association for multiple new genes of which some are known for their role in HDL-cholesterol metabolism such as ABCG1 or GPR109A.

Centriolar satellites are assembly points for proteins implicated in human ciliopathies, including oral-facial-digital

syndrome. Lopes CA et al. J Cell Sci. 2011;124:600-12.Ciliopathies are a collection of developmental and degenerative single-gene disorders characterized by the dysfunction of a hair-like organelle called the cilium. Cilia can be found on the surface of most mammalian cells explaining why cilia-related disorders can affect a wide range of organ systems. Cilia originate from a basal body which can be seen as a modified centrosome. In this study, Professor Andrew Fry’s group in the Department of Biochemistry, used fluorescence microscopy to show that OFD1 (involved in oral-facial-digital syndrome 1), BBS4 (involved in Bardet-Biedl syndrome) and CEP209 (involved in Joubert syndrome), colocalize with PCM-1. PCM-1 is a key component of structures called centriolar satellites, particles surrounding basal bodies and centrosomes and involved in centrioleassembly. Depletion of any of these four proteins affects the localization to the centriolar satellites of the other proteins. This might give an explanation why the clinical spectrum of these syndromes overlap.

Here, we report a selection of recent articles which at some point have involved our equipment or our expertise. We are pleased to see that our users have been successful in publishing articles in journals with good impact factors. Visit Genomics Core Facility and Advanced Imaging Facilities for the full list of publications.

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MicroRNA expression profiling in patients with lamin A/C associated muscular dystrophy. Sylvius et al., Faseb J.2011 In Press.Laminopathies are a group of disorders caused by mutations in the gene coding for the lamin A/C, a protein of the nuclear envelop. Dr Sylvius with Dr Shackleton’s group (Depatrment of Biochemystry) and the “Institut national de la santé et de la recherche médicale” in Paris have demonstrated that the microRNAtranscriptome is dramatically affected in patients with lamin A/C related muscular dystrophy. The group has been able to identify several microRNA species dysregulated in this disease and decipher their function thus providing new insight in the pathophysiological mechanisms of the disease. This study provides a good example of a productive interaction between the Genomics Core and the Advanced Imaging facilities. Sylvius and colleagues first performed a whole-genome microRNA transcriptome analysis in patients affected by this disorder using the qPCR systems available in the Genomics Facility. To better understand the function of these microRNAs found dysregulated in these patients, they then performed loss- and gain-of function analyses and cell proliferation assays using the

Olympus ScanR screening station.

The kynurenine pathway modulates neurodegeneration in a Drosophila model of Huntington's disease. Campesan et al. Curr Biol. 2011 Jun7;21(11):961-6.Huntington’s disease (HD) is an incurable, inherited, neurodegenerative disease characterised by involuntary movements and memory loss. Using a fruit fly model of Huntington disease, Dr Giogini’s group from the department of Genetics, has demonstrated that the inhibition of two major components (the Kynurenine 3-monooxygenase and the tryptophan 2.3-dioxygenase ) of the kynurenine pathway which is responsible for tryptophan degradation, can increase the level of kynurenic acid, a neuroprotectivemetabolite and thus modulate neurodegeneration

Automatic cell counting and cell cycle analysis on the olympus ScanR.

Recent studies and publications from users of the facilities

E x p r e s s N e w s

•The Genomics Core Facility and the Advance Imaging facilities are continuously trying to stay up-to-date with new technologies. If you think there are new pieces of equipment we should acquire or new services we should develop, your suggestions and ideas are welcome. We will be more than happy to see if they can be beneficial for other researchers of the university and then identify the possible funding to implement them within the facilities. Feel free to email us or come and discuss with us.

•Ho w to save on the cost of microarray and next-generation sequencing experimentsAlthough next generation sequencing or microarray experiments are still quite expensive, there are ways to cut down the cost of such experiments. Sample pooling is one of them, especially in gene expression studies. However, this option is not always possible depending on the type of experiment. If you have a small budget and a study involving just a few samples, another alternative is to share runs with other users of the platform. All you have to do, is let us know about your number of samples and your needs, so we can put you on a waiting list. As soon as we have other users with similar needs, we will pair you up with them and will ask you to share the cost. At the Genomics Facility, we have already accommodated several “mixed” projects like this. Providing that you are not in the rush, this solution proved to be a cheap way to access these technologies.

•Reduced prices or free access to CBS facilitiesThe Advance Imaging Facilities (AIF) are part of the Centre for Core Biotechnology Services (CBS). We charge for use of equipment or services within CBS to cover the running and maintenance costs. However, if you have no funding but need access to a CBS facility, for example to obtain preliminary result or finish some research, please fill out the proforma form .The AIF has become a small research facility in February 2010 and can be found under 'Research facilities' in Lucre for grant applications.

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E x p r e s s N e w s

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•Next Generation Sequencing at LeicesterThe Roche/454 platform is being utilised for different applications for researchers both within and outside the University. •Continuous developments with this platform have meant that researchers have had access to the latest in sequencing technology on campus. To find out more about applications, experimental formats, options, and prices, contact Reshma Vaghela (rjb40@le.ac.uk) or see our website.

Access to the technology is open to all, and no matter how large or small your project is, we can accommodate projects to offer cost effective sequencing. We are currently accepting samples for forthcoming experiments!

If you are interested in the latest Roche/454 technology developments, which now mean you can obtain even longer, Sanger-like read lengths using this technology, (85% of total bases from reads longer than 500 bp; longest reads are over 1 kb in length)....please get in touch so we can see what the need is for the latest upgrade to our system here at Leicester.

List of Equipment

Illumina Bead Station microarray platform : High throughput genotyping, gene expression, methylation analysis

Roche/454 GSFLX: Next-generation sequencing system

DASH: Robust, low/medium throughput SNP genotyping that reveal CNVs

Roche LightCycler 480 real-time quantitative PCR system with 96-well and 384-well capacity

ABI 7900 HT real-time PCR system with 384-well capability, Taqman Low Density Array (TLDA)

Pre-PCR Clean room: Fully furnished, dedicated room for PCR set up

Bioruptor Sonicator: Precision sonicationsystem

Hydroshear: Uses hydrodynamic shearing forces to fragment DNA strands

Genepix Micro-array Scanner: 3 colour system with sample autoloader

Biomek FX Robot: General purpose liquid handling device, with many add-ons

Agilent Bioanalyser: For accurate quantification and quality control of DNA, RNA and protein

Nanodrop 8000: For measuring absorbance of DNA, RNA, dyes, proteins & microbial cell culture OD – 8 samples measured at once

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Platforms and instruments currently available in the Genomics Facility:

List of Equipment

Systems available within the AIF

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Microscope systems

2 inverted Nikon TE300 microscopes each equipped with an Hamamatsu ORCA-R2 digital camera and an X-cite120 fluorescence illumination system.

Nikon Eclipse Ti-E microscope with LEDs and an Andor iXon EM-DU897 EM-CCD camera

Leica TCS SP5 confocal laser scanning microscope

Olympus CellR/ScanR imaging system for live cell imaging and screening

Olympus FV1000 confocal laser scanning microscope

Olympus upright BX61 automated microscope with Applied Spectral Imaging camera and software for spectral imaging, karyotyping and automatic spot finding

Nikon eclipse Ti microscope for TIRF (soon also with LEDs)

Zeiss MP7 multi photon laser scanning microscope

Olympus LV200 bioluminescence imaging system

Nikon C1Si confocal laser scanning microscope (including spectral detectors)

Non-microscope systems

BD FACSCanto II

GE Typhoon Trio+ imager

Fujifilm LAS4000 luminescence imager

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Page 2. Picture from “the Nutty Professor” by Jerry Lewis

Members of staff

Genomics Core FacilityProf Anthony J Brookes

Dr Nicolas SylviusMrs Reshma Vaghela

Advanced Imaging FacilitiesDr Kees R. Straatman

Miss Tara HardyMr Matt Barker

Miss Shashi Rana