Quantitative measurement of calretinin and β-actin mRNAIN rat brain micropunches without prior isolation of RNA
Abstract 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 (10–500 μ protein). 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 treated with ribonuclease in a single tube. We have determined the mRNA levels of calretinin, a predominantly neuron-specific calcium binding protein in microdissected nuclei and other regions of rat brain. For interassay comparison, measurement of sample protein and β-actin mRNA permits normalization and quantitation in terms of these internal controls. The quantity of calretinin mRNA ranged from 281 ± 35 fg/μg protein in the thalamic paraventricular nucleus to 2.3 ± 0.5 fg/μg protein for the cerebral cortex. The calretinin/β-actin ratios ranged from 79.9 ± 9.3% to 1.3 ± 0.1%, respectively. The combination of microdissection techniques with a lysate RNase protection assay: (1) establishes this technique as quantitative for detection of high and low abundance mRNAs from microdissected brain specimens; (2) bypasses the inefficiencies and uncertainties associated with isolating RNA; and (3) enables large numbers of determinations from discrete brain nuclei to be analyzed in 2 to 3 days.