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JoVE Journal

Genetics

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Saccharomyces cerevisiae Metabolic Labeling with 4-thiouracil and the Quantification of Newly Synthesized mRNA As a Proxy for RNA Polymerase II Activity

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09:21 min

October 22nd, 2018

October 22nd, 2018

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0:04

Title

0:41

Cell Culturing

1:37

4tU Labeling with S. pombe as a Spike-in

3:20

RNA Extraction and DNase Treatment

4:30

Thiol-specific Biotinylation of Newly Synthesized RNA

6:05

Purification of Newly Synthesized Fraction from Total and Unlabeled RNA Using Streptavidin-coated Magnetic Beads

7:18

Results: Quantification of Newly Synthesized mRNA as a Proxy for RNA Polymerase II Activity

8:52

Conclusion

필기록

The main advantage of this technique is that it reflects RNA polymerase II activity by measuring newly-synthesized mRNA which are not influenced by RNA degradation. This protocol was originally described using microarray hybridization, but can eas

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The protocol described here is based on the genome-wide quantification of newly synthesized mRNA purified from yeast cells labeled with 4-thiouracil. This method allows to measure mRNA synthesis uncoupled from mRNA decay and, thus, provides an accurate measurement of RNA polymerase II transcription.

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