Table 20.1.

Methods for measuring mRNA half-lives in yeast

Method		Advantage	Disadvantage
In vivo labeling	Approach to   steady-state   pulse-chase	Minimal cell perturbation No need for special strain or   construct Can monitor the half-life of many   mRNAs simultaneously	Requires large amounts of radioactive   material Poor signal-to-noise ratio for mRNA   of low mRNA abundance
Transcriptional   inhibition	With drugs   (thiolutin, 1,10-   phenanthroline)	No need for special strain or   construct Can monitor the half-life of many   mRNAs simultaneously	May cause a loss of labile factors May alter the decay of specific   mRNAs May alter other cellular pathways   (i.e., transcription or translation)
Transcriptional   inhibition	Temperature-sensitive   RNA polymerase II   mutant (rpb1-1   mutation)	No need for special construct   Can monitor the half-life of many   mRNAs simultaneously	May cause a loss of labile factors Not useful with other conditional   mutants Possible secondary complications   caused by heat shock Requires a special strain
Transcriptional   control	Regulatable GAL   promoter	Minimal cell perturbation Applicable to transcriptional   pulse–chase experiments	Requires a special construct Allows only mRNAs under control of GAL promoter to be analyzed Changing the carbon source may alter   mRNA stability
Transcriptional   control	Regulatable   ‘‘Tet-off’’	Minimal cell perturbation Applicable to   transcriptional   pulse–chase experiments Allows an accurate control   of inhibition or induction	Requires a special construct Allows only mRNAs under control of   Tet-promoter to be analyzed The antibiotics used may alter cellular   metabolism, influencing the gene   under control