| Reference |
Wang Y, Liu CL, Storey JD, Tibshirani RJ, Herschlag D, Brown PO. Precision and functional specificity in mRNA decay. Proc Natl Acad Sci U S A. 2002 Apr 30 99(9):5860-5 (free online article) supporting site, please see primary sourcePubMed ID11972065
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| Method |
By using DNA microarrays, researchers precisely measured the decay of each yeast mRNA, after thermal inactivation of a temperature-sensitive RNA polymerase II: Yeast was grown on YPD at 24 degrees celsius, transferred to a similar medium at 49 degrees and 0, 5, 10, 15, 20, 30, 40, 50, and 60 min after the temperature shift harvested on a nitrocellulose filter followed by liquid nitrogen freezing and total RNA extraction by hot phenol extraction. A nonlinear least squares model was fit to determine the decay rate constant (k) and half-life (t1/2) of each mRNA. The decay rate constant, k, is the value that minimized Si = 1,n[y(ti) - exp(-k×ti)]^2, where y(t) is the mRNA abundance at time t and the summation is taken over all observations for the particular mRNA. The half-life is t1/2 = ln2/k. The goodness of fit of the decay model for each gene was assessed with the F statistic (ref 20 in article), based on the null hypothesis that the data fit a first-order decay model.
A bootstrap method was used to calculate confidence intervals for both t1/2 and k (ref 21 in article). To examine the global relationship between poly(A) shortening
and mRNA turnover, researchers made a separate series of measurements
of the fate of each mRNA, using an anchored
oligo(dT) primer (5'-T20VN-3'), rather than random primers, in
the cDNA probe synthesis. This approach allowed them to track
specifically the mRNAs that retained poly(A) tails of sufficient
length to allow priming. The poly(A)+ mRNA decay half-lives,
as defined in their assay, were distributed within a narrower range
and were significantly shorter (peak at 10–15 min) than the
overall mRNA decay half-lives (Fig. 2A). |