Tissue preparation and banking

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Abstract

With the increasing application of genomic and proteomic technologies to the research of neurological and psychiatric disorders it has become imperative that the postmortem tissue utilized be of the highest quality possible. Every step of the research design, from identifying donors, acquiring sufficient information for accurate diagnosis, to assessing tissue quality has to be carefully considered. In order to obtain high-quality RNA and protein from the postmortem brain tissue a standardized system of brain collection, dissection, and storage must be employed and key ante- and postmortem factors must be considered. Reliable RNA expression and protein data can be obtained from postmortem brains with relatively long postmortem intervals (PMIs) if the agonal factors and acidosis are not severe. While pH values are correlated with RNA integrity number (RIN), a higher pH does not guarantee intact RNA. Consequently RNA integrity must be assessed for every case before it is included in a study. An analysis of anti- and postmortem factors in a large brain collection has revealed that several diagnostic groups have significantly lower pH values than other groups, however, they do not have significantly lower RIN values. Moreover, the lower pH of these groups is not entirely due to agonal factors and/or smoking, indicating that these subjects may have additional metabolic abnormalities that contribute to the lower pH values.

Introduction

The development and increasing application of genomic and proteomic technologies to the research of neurological and psychiatric disorders has made it imperative that the postmortem tissue utilized be of the highest quality possible. Every step of the research design, from identifying donors, acquiring sufficient information for accurate diagnosis, to assessing tissue quality has to be carefully considered. In this review, we will briefly outline the steps necessary to acquire useful brain tissue. Many of these steps have been reviewed in great detail in other publications (Kleinman et al., 1995; Vonsattel et al., 1995; Green et al., 1999; Kittel et al., 1999; Shankar and Mahadven, 1999; Lewis, 2002; Bunney et al., 2003; Hynd et al., 2003; Katsel et al., 2005) and so will only briefly be discussed here. Demographic factors that affect the integrity of the tissue, and the RNA in particular, will be discussed in more detail.

Section snippets

Recruitment

Brain donations are generally obtained by direct donations from individuals affiliated with patient advocacy groups, from hospices, from state psychiatric hospitals or veteran's administration (VA) hospitals or from medical examiner's offices. Each source has advantages and disadvantages. For example, donations through patient advocacy groups allow accurate diagnosis of living subjects but the tissue is obtained from multiple sources, and often harvested with nonstandardized techniques. Brain

Handling tissue

It is important to have all tissue for any one study collected in the same manner, e.g. freezing, dissection, and the preparation of samples. All personnel involved in the removal of the brain and the processing of it should be trained centrally by the same team to ensure that each specimen is handled in exactly the same manner.

Freezing/fixing

A commonly used protocol is to have the cerebrum hemisected, one hemisphere fixed in formalin and the other cut into 1 cm thick coronal slices and frozen in an

Demographic database

Each case is assigned a number and entered with all relevant variables into a demographic database. Examples of variables include age, race, sex, date of birth, date of death, cause of death, rate of death, refrigeration interval, postmortem interval, pH of brain, RNA quality (e.g. RNA integrity number (RIN) number), left brain (frozen or fixed), brain weight, weight of deceased, and height of deceased. Other variables deemed necessary to include would depend on the nature of the diagnostic

Factors affecting RNA integrity

The emergence of genomic technologies requires that intact RNA be recovered from postmortem specimens. As each brain enters the collection, RNA is extracted and evaluated. If the RNA is degraded it may not be an efficient use of resources to further process the tissue and retrieve and analyze the medical records. The Stanley Medical Research Institute (SMRI) Laboratory has assessed total RNA quality on over 450 cases using the RIN determined by electrophoresis using the Agilent 2100 Bioanalyzer

Protein integrity

Although a considerable number of publications have appeared on the proteomic analysis of human postmortem brain samples (Edgar et al., 1999a, Edgar et al., 1999b; Greber et al., 1999; Edgar et al., 2000; Johnston-Wilson et al., 2000; Schonberger et al., 2001; Butterfield and Castegna, 2003; Lubec et al., 2003; Butterfield, 2004; Lewohl et al., 2004; Prabakaran et al., 2004; Swatton et al., 2004; Newcombe et al., 2005) surprisingly little is known about quantitative postmortem changes in the

Conclusions

High-quality RNA and protein can be extracted from postmortem brain tissue provided a standardized system of brain collection, dissection, and storage is employed and key ante- and postmortem factors are considered. Reliable RNA expression and protein data can be obtained from postmortem brains with relatively long PMIs if the agonal factors and acidosis are not severe. While pH values are correlated with RIN values, a high pH does not guarantee intact RNA, consequently RNA integrity should be

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