MOLECULAR AND CELLULAR IMAGING CORE

Aims:

• To provide state-of-the-art facilities for qualitative and quantitative film densitometry and microscopic tissue analyses for investigators in the field of aging research.

Introduction:

Diseases associated with aging may have characteristic alterations in a tissue such as the amyloid plaques and neurofibrillary tangles found in the brains of individuals with Alzheimer's disease. Specific subtypes of cells may be lost in diseases associated with aging such as dopaminergic neuron loss in Parkinson's disease. Alterations in gene and protein expression occur in a variety of age-related diseases, organs and specific cells within organs. Understanding the mechanism for these tissue changes requires qualitative and quantitative assessment of tissue sections fom both people with an age-associated disease and from animal models of these diseases. Tissue section assays include hisologic stains, immunohistochemisty (IHC), quantitative receptor autoradiography (QAR), and in situ hybridization histochemistry (ISHH) among others. The Molecular and Cellular Imaging Core (MCIC) provides facilities and expertise for qualitative or quantitative analysis of tissue sections used by researchers in the field of aging. The availability of equipment and technical assistance attracts investigators to these powerful techniques which might otherwise have been considered too challenging, time consuming, or costly to pursue. The ability to use standardized methods of analysis and standardized probes for ISHH reduces the amount of effort required by investigators using these techniques. Common techniques and probes will also enhance comparisons and discussions among investigators.

Facilities and Services:

The Molecular and Cellular Imaging Core (MCIC) provides the following facilities and services.

(1) Performance of selected tissue-based assays including quantitative receptor autoradiography (QAR) or in situ hybridization histochemistry (ISHH) for selected projects or faculty.

(2) Development of new tissue-based assays including QAR and ISHH probes for selected projects or faculty based on a review of requests and available resources.

(3) Development of species and tissue specific ISHH probes in conjunction with the Microarray Analysis Core (MAC) that can be used as control or comparison probes by multiple investigators.

(4) Assist individuals in developing in situ hybridization probes for verification and secondary analyses of data obtained from the MMC.

MCIC Technical Assistance:

Technical assistance is provided by the MCIC. Advice on assays for a given project is also provided. A form for questions to query the core via Email is available to investigators. Certain QAR, IHC, or ISHH assays are performed by the core for users. One half-time technical assistant staffs the MCIC and/her time is allocated to a variety of functions. Design of new assays or probes is the responsibility of the core director along with the technical assistant. The technical assistant is available for questions and tutorials as time permits. In situ hybridization histochemistry is performed as part of the MCIC activities as discussed below in collaboration with the MAC.

Collaboration with Microarray Analysis Core (MAC):

It is necessary for users of the MAC to confirm and extend their initial findings using a complementary and cell-based method. Confirmation of major changes in gene expression must be demonstrated not to be an artifact of sample preparation or selection bias. Extension of initial findings using additional samples, cases, regions, or conditions depending on the experiment is required. The limited number of samples that can be processed using arrays and the expense of arrays requirse users to consider alternative methods of analysis and quantifying of gene expression. Among the many ways to measure gene expression using RNA measurements (Northern blotting, ribonuclease protection assays, and RT-PCR) in situ hybridization histochemistry has a number of distinct advantages. The amount of tissue needed for hybridization is small; i.e. an area as small as 0.5 cm x 0.5 cm x 10 µm can be used for hybridization. Multiple tissue sections can be taken from a tissue at one time, stored at Ð70 degrees C, used at will, and hybridized to a number of different probes. Multiple tissue sections from different samples can be processed at the same time. Anatomical resolution for different regions is excellent (particularly for the brain) and is limited by the resolution of a camera or microscope. Finally, cellular localization can be determined using emulsion autoradiography or nonisotopic methods combined with microscopic imaging. Given these advantages, we believe ISHH may be the method of choice for confirming data obtained from gene arrays using tissue samples. ISHH can be a difficult technique for inexperienced users to initiate. The existance of a core to perform or assist in this is a major benefit to users. If the expression level of a particular gene is moderate to high, oligonucleotide probes are ideal for use in ISHH. They are easy to design, can be designed to be specific for a particular member of a gene family, are inexpensive to make and label, and easy to process. The MCIC provides a number of services that benefit users of the MAC. The MCIC develops and validates a set of (initially) five oligonucleotide housekeeping probes selected in conjunction with the MAC director. Probes to the identical housekeeping genes, but specific for different species will be made. Probes will be for genes contained on current gene chips and custom arrays. Initial genes include §-actin, GAPDH, cyclophilin, transferrin receptor, and elongation factor 1a.. These were selected based on their levels of expression, ubiquitous expression in multiple tissues and cells, known sequence, and common usage by investigators as controls in a variety of RNA techniques. Experience using the same genes across different species and tissues provide us with a database of values that are useful as we begin to understand gene expression arrays and their validation by ISHH or other methods. Correlations between the absolute values and the ratios between different housekeeping genes obtained from gene arrays and from ISHH will allow us to predict values from one technique to the other. In other words, with experience we will be able to use the values obtained from a gene array to determine if an oligonucleotide probe will be sensitive enough to detect and quantify the mRNA level. It also allows us to predict how long we will need to expose slides to film or emulsion to detect an appropriate signal. If the level of expression is too low for oligonucleotide probes, we can assist users in the development of longer RNA probes of higher specific activity. This method is more labor intensive and may involve cloning, restriction digests, plasmid purification, and in vitro transcription. This method is not amenable to examination of more than a few genes suspected to be of biological importance. It is likely to be used for analysis of genes that might be down regulated in a biological process.