Stability of brain RNA post rnortern : effect of Alzheimer ' s disease

Many neurological and psychiatric disorders are genetic (e.g. Huntington's chorea), have a genetic component (e.g. Alzheimer's disease, bipolar depression) or are thought to involve alterations in gene expression (e.g. Pick's disease, Alzheimer's disease). It would, therefore, be useful to examine gene expression in post-mortem human brains. For this reason we have assessed the stability of brain RNA with post-mortem delay using sheep tissue. We have also isolated RNA from normal and Alzheimer human brain and determined its yield and integrity. Total and mRNA was prepared from sheep brains at 0,4, 8,24 and 48 h post mortem. Before use the brains were stored so as to mimic post-mortem cooling conditions of human brain awaiting autopsy, and then frozen rapidly in liquid nitrogen. Total RNA was prepared by the guanidinium thiocyanate/CsCl method (Chirgwin et al., 1979) and mRNA by one passage of total RNA over an oligo(dT)-cellulose column. The amount of total RNA, as estimated by absorbance at 260 nm, remained unchanged up to 24 hpost mortem and decreased to 50% of the fresh value from 24 to 48 hpost mortem (Fig. la). The yield of mRNA expressed as a percentage of the total remained unaltered with post-mortem delay (Fig. 16). Agarose gel electrophoresis and ethidium bromide staining of both total and mRNA showed no change in profile up to 48 h post mortem. The rate of in vitro protein s nthesis of mRNA as measured by the incorporation of [ Hlleucine was found to be similar for all mRNA samples of differing post-mortem delays (Fig. 1 c) . Autoradiographs of one-dimensional polyacrylamide gels indicated that high molecular mass proteins (at least up to 100 kDa) were synthesized (Laemmli, 1970; Marrota et al., 1981). Total and mRNA was prepared from six normal human brains (post-mortem delay 7.3 f 2.25 h, age 70 f 6.4 years). The yield of total RNA was 167.5 f 17.7pgIg of starting tissue (slightly less than that of sheep). The percentage of mRNA obtained from total RNA (5.58 f 1.2%) was within the normal range. This human RNA was electrophoresed in a denaturing gel containing 1.5% agarosel2.2 Mformaldehyde (Lehrach et al., 1977; Goldberg, 1980). The gel was blotted to hybond nylon (Amersham) (Southern, 1975) and fixed by U.V. irradiation for 5 min. The Northern blot was probed with a '2P-labelled cDNA actin probe (Minty et al., 1981). This probe detected a single band at 2 kb, indicating that the RNA is in an undegraded form. Agarose gel profiles of human RNA were similar to those of sheep. Translation of this human mRNA in vitro gave similar rates of protein synthesis to those found when using sheep brain. To summarize, these data indicate that intact and biologically active mRNA can be isolated from post-mortem brain for at least 24 h post mortem. This suggests that it should be possible to study differential gene expression in health and disease. To this end total RNA and mRNA was prepared from six Alzheimer brains (age 70 f 6.4 years, post-mortem delay 10 f 3 hours). The yield of total RNA (1 37 f 13 pg/g of starting tissue) was similar to that of the normal human brains and the yield of mRNA (3.3 f 0.5%) was slightly less compared with the normal brains, but was within the normal range and similar to the results obtained using sheep tissue. These preliminary studies indicate that the yield of total RNA is the same in both control and Alzheimer brains, but suggests that the yield of mRNA is slightly lower in Alzheimer brain (Sajdel-Sulkowska et a[., 1983, Taylor et al., 1986). Y i