MOLECULAR BIOLOGICAL STUDIES OF CALRETININ

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A microdissection technique for quantitation of neurochemicals in discrete brain nuclei has been applied to quantitative measurement of mRNA. The method permits quantitation of low abundance mRNA from submilligram amounts of tissue. Discrete nuclei and other regions of the brain are solubilized in concentrated guanidine thiocyanate solution, mRNA is directly hybridized with riboprobes, and detected with a ribonuclease protection assay. This method eliminates the necessity for RNA isolation from solid tissue. No assumptions regarding RNA recovery are necessary since tissue specimens are solubilized, hybridized and nucleased in a single tube. Simultaneous detection of mRNAs of interest, with total sample protein and the abundant 'housekeeping' gene (3-actin, permits normalization and quantitation in terms of these internal controls. We have determined the levels of calretinin, a central nervous system neuron-specific calcium binding protein and beta-actin in microdissected nuclei and other regions of rat brain. The quantity of calretinin mRNA ranged from 281 +/- 35 fg/mg protein in the thalamic paraventricular nucleus to 2.3 +/- 0.5 fg/mg protein for the cerebral cortex. The calretinin/beta- actin ratios in the same specimens ranged from 79.9 +/- 9.3% to 1.3 +/- 0.1%, respectively. Our modifications to the lysate RNase protection assay: (1) bypass the inefficiencies and uncertainties associated with isolating RNA from microdissected brain specimens; (2) establish this technique as quantitative for detection of high and low abundance mRNAs; and (3) enable large numbers of determinations from multiple brain nuclei to be analyzed in 2 to 3 days.