Describe 3 ways of measuring changes in mRNA expression and provide an explanation of how each one works. Does the method measure mRNA synthesis, degradation, or levels?
1. Nuclear run-on assay: Measures relative amounts of mRNA synthesis.
a. Isolate nuclei (keep cold to inhibit transcription) at the different times being examined (e.g., lengths of treatment).
b. Add 32P-labeled rNTPs, allow transcription of nascent RNAs to continue.
c. Hybridize RNA to complementary DNA of interest.
d. Measure level of radioactivity of the DNA-RNA hybrids