Elsevier

Methods in Enzymology

Volume 152, 1987, Pages 633-648
Methods in Enzymology

[67] A molecular titration assay to measure transcript prevalence levels

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    The RNA was then quantified by measurement of absorbance at 260 nm. The abundance of the mRNA encoding for gephyrin in the total RNA extract was measured by the RNase protection assay (Lee and Costlow, 1987; Zinn et al., 1983), which was performed as described by Follesa et al. (1998). Total RNA (25 μg) was dissolved in 20 μl of hybridization solution containing 150,000 cpm of the 32P-labeled cRNA probe (specific activity, 6 × 107 to 7 × 107 cpm/μg) encoding for gephyrin, and 15,000 cpm of the 32P-labeled cRNA probe (1 × 106 cpm/μg) encoding for cyclophilin (Danielson et al., 1988; Milner and Sutcliffe, 1983), that was used as an internal standard for our measurements (Follesa et al., 1998).

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    An example of an absolute kinetic gene expression measurement is shown in Fig. 3. The internal standard most used for this purpose in our lab is the mRNA of the gene SpZ12.1, the abundance of which was quantified by single strand probe excess RNA titration (Lee and Costlow, 1987; Wang et al., 1995). One useful aspect of a thorough knowledge of the temporal gene expression profile is that it affects the choice of timepoints to be analyzed in perturbation experiments.

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