The QMB Core will facilitate the quantification of mRNA and DNA requirements for faculty and staff. This core also offers access and consultation to technologies designed to characterize and quantify changes in gene expression. The QMB core is located at the MSC and adjacent to the University of Wisconsin Biotechnology Center, which is the location of the facilities required for the microarray analysis.

Services

Primer Design

Investigators unfamiliar with PCR primer design can consult with the QMB CORE to obtain the primers best suited for PCR needs, including specialized primers for nested PCRs and primer design with restriction sites for sub-cloning. Primer design strategies to amplify homologous sequences from species where there may not be a published target sequence are utilized. Design criteria are now established to utilize MIQE guidelines.

PCR optimization

PCR will be performed across a temperature gradient of 40-70°C using an Eppendorf epgradient Mastercycler (acquired 2003). Products will be analyzed by standard agarose gel electrophoresis, followed by ethidium bromide staining, and a digital image will be provided to the user. The user will provide the primers and template DNA for this service unless “Full Service” is requested.

Quantitative real time PCR (qPCR)

Optimized qPCR will be performed on an ABI Quant Studio 7 Flex RT PCR or  ABI 7300 RT PCR machines using Taqman SYBR Green I (as the primary dye). Data generated from each run will be exported into an Excel Spreadsheet for use by the customer. The customer may also be able to utilize the ABI software on the 7300 machine for analysis provided there is no run ongoing. ABI analysis software is also available for use on remote computer stations, but this is not a service that is available at this time. The fragments generated in each qPCR reaction can also be independently analyzed by agarose gel electrophoresis upon request.

Each qPCR run will also contain data on meltcurves for each reaction vial, which provides information on the specificity and integrity of the product bands in the sample (low stringency products, such as primer dimers, are detected using this feature).

Although SYBR Green is the dye choice available for this service, the 7300 also has the capacity to detect multiple dyes allowing for multiplexing of PCR products. Users interested in multiplexing should be prepared to purchase their own reagents separately rather than use Core reagents for a fee.

Four services for qPCR are available, including:

• Run Only, where the User prepares a plate or strip tubes of samples and reagents from his/her own laboratory.

• Setup and Run, where the User brings in template DNA and primers. The Core facility will prepare each reaction accordingly and execute the PCR run.

• Full Service, where the User brings in sample RNA (and primers) only. The Core facility will prepare the template cDNA, optimize the PCR conditions, and execute the qPCR run.

• Micro qPCR array for mouse retinal ganglion cell gene expression. The QMB CORE provides a specialized service to assay the expression profile of a subset of genes expressed in retinal ganglion cells using a MIQE-conforming mini-array. Each 96 well plate contains the capacity to assay two separate samples each for the expression of 15 different genes, including 8 genes normally expressed in healthy ganglion cells, 4 stress response genes, and 3 house-keeping genes. Each array also contains a base-line standard curve to enable the user to quantify molecules of target transcripts per sample and perform relative quantitative analysis between samples. Specifics of each transcript is available on request. Each plate has the capacity for 2 different samples per run.

Probe synthesis

RNA antisense (and sense control) probes (riboprobes) will be synthesized upon request. Probe synthesis can be made using radioisotopic incorporation, or nonradioactive probes can be synthesized using digoxigenin labeled nucleotides. Probe synthesis will be conducted using constructs of target sequences cloned into appropriate plasmid vectors that contain RNA polymerase binding sites flanking the target sequence. Plasmid should be provided (unless otherwise contracted) complete with a restriction map. The Core will linearize and purify the plasmid, and then make and purify riboprobes ready for use.

The user has several options for this service including:

• Digoxigenin labeled probes – RNA probes will be made by incorporating 11DigUTP into the reaction. Probes will be purified by eliminating template DNA and phenol chloroform extraction and precipitation. Purified probe will be assayed by formaldehydegel electrophoresis.

• Radioactive probe synthesis – RNA probes will be synthesized by incorporation of radioisotopes into the synthesis reaction. For 32PUTP labeled probes used for RNase protection assays, probes will be synthesized and purified by elution from urea polyacrylamide denaturing gels.

• Full service probe preparation – Full service preparation will include initial cloning (or subcloning) a suitable target sequence into a plasmid that can be used for riboprobe synthesis. This service can include PCR cloning of a region of a target molecule, or subcloning of a small fragment from a larger DNA sequence provided by the user. Cloning or subcloning strategies (to achieve the most optimum target sequence for riboprobes, depending on the application) will be initially conducted in consultation with the Core director.

Allelic discrimination of mouse genomic DNAs

Screening of mouse genomic DNAs will be conducted using single nucleotide polymorphism (SNP) TaqMan genotyping technology. Samples will be analyzed on the ABI 7300 qPCR machine using preformulated or custom primer sets from ABI, which will be provided by the user. PCR runs of up to 96 samples per run will be analyzed using ABI allelic discrimination software and sample identity will be provided as an Excel print out. If the user has samples involving mouse strains that require custom oligo preparation, the CORE offers the service of testing conditions for each custom set using control DNAs provided by the user.

Quantification of promoter transcription activity by reporter gene analysis

The QMBC now offers instrumentation to monitor Luciferase reporter gene activity for transient transfection assays or for assays from transgenic mice carrying appropriate reporter genes. Assays can be read singly in a Turner Model TD-20e Luminometer or multiple-plate samples can be read in a Tecan Safire2 multiplate reader. Both instruments are capable of reading firefly and Renilla Luciferase for the Duo-Glo gene reporter system. Users are expected to provide plates, tubes and reagents for use in the machines.

Proteomics

The core can either assist users in running 1d or 2d gels or run the gels for the users. The core also serves as the liason between users and the HPLC and Mass Spec facility at the UW-Biotechnology Center for the analysis of bands or spots in gels and probes, and consulting for appropriate measures to use in analysis samples. Finally, the core provides expertise in identification of phosphorylation sites.

Consultation

Consultation services area available on all services rendered by the QMBC upon request.

Special Services

In special circumstances, the QMB Core can provide full services to quantify RNA samples using conventional methods such as Northern Blotting and RNase Protection Assays (RPA).

Northern blotting services can be obtained in two formats, including a full service format where the QMBC will run formaldehyde gels of total RNA or polyA(+) mRNA (provided by the User), blot the RNA to GeneScreen Plus nylon membrane, and probe the membrane with probes made by the QMB Core. Probed filters will be exposed to Xray film or PhosphorImager screens.

A secondary service provided by the QMB Core will be to run the formaldehyde gels and blot the RNA to membrane. The membrane will then be provided to the User.

RPA can also be contracted out using the QMB Core. RPA is done using total RNA provided by the User and probe prepared by the QMB Core. Digestions are done using a method employing RNase ONE as the digesting nuclease. The QMB Core will expose the resulting digests to X-ray film and quantify the protected fragments using PhosphoImager analysis.

Special services are provided only when there is available time in the QMB Core or in certain circumstances. Consultation with the QMB Core is strongly recommended if these services are desired.

Services under development

A new service under development is a high through-put analysis of neurite outgrowth from retinal explants.

A major event in the pathogenesis of glaucoma pathology is the damage and autonomous destruction of ganglion cell axons in the optic nerve. This event is likely the earliest damaging event to the retinal ganglion cells, and probably initiates the apoptotic degeneration of ganglion cell somas in the retina. Since the majority of neuroprotection strategies are directed at preventing soma loss, and are not focused on axon regeneration, technologies that encompass the ability to assess both are warranted.

In this high-throughput assay, we embed retinal explants in a collagen matrix. Explants allow for intact cellular interactions because retinal architecture is preserved, and is preferable to assays using isolated ganglion cells. Numerous explants can be generated from a single retina (up to 8 per retina from a single mouse), allowing for testing of multiple treatment groups from tissue with the identical genetic background.

Also, we are developing 3 types of explants. The first is using PN7 retinas (mouse) in which ganglion cells still retain developmental growth potential. These experiments are suited to assess media conditions that promote a growth permissive and stimulatory environment. The second is explants from adult eyes, which tests environments that promote survival and enhanced growth stimulatory environments. The third is adult explants from Bax-/- mice, in which apoptotic ganglion cell death has been blocked. This tests for environments that stimulate axonal regeneration from adult cells without confounding the experiment with a cell survival/cell loss component.

Outgrowth, which is predominantly axonal, can be measured continuously in the same explant in longitudinal studies. At the end of a select time point, the number, length, and branching of neurites can be assayed from explants stained with phalliodin. Digital images are then assessed using an adaptation of MatLab software that has been imported into the facility.

For questions regarding Quantitative Molecular Biology services, please contact:

QMB Core Director

Robert W. Nickells, Ph.D.
571A Medical Sciences
1300 University Ave.
Madison, WI 53706
Phone: 608-265-6037
Email: nickells@wisc.edu