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Northern Blot Protocol v1
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Northern blot is a hybridization technique to visualize and quantify RNAs from cells or tissues using a radio-labeled probe. mRNAs only constitute a small fraction of total RNA in cells. Through enrichment of poly(A)-tailed mRNAs, this protocol provides increased sensitivity for the detection of lowly expressed mRNAs. In addition, RNA denaturation is achieved using glyoxal (in contrast to formaldehyde-based methods), resulting in sharper bands. We have used this protocol extensively to detect and quantify the expression of different 3'UTR isoforms originating from the same gene 1-5. 1 Mayr, C. and D. P. Bartel (2009). Widespread shortening of 3'UTRs by alternative cleavage and polyadenylation activates oncogenes in cancer cells. Cell 138(4): 673-684. 2 Lianoglou, S., V. Garg, J. L. Yang, C. S. Leslie and C. Mayr (2013). Ubiquitously transcribed genes use alternative polyadenylation to achieve tissue-specific expression. Genes Dev 27(21): 2380-2396. 3 Berkovits, B. D. and C. Mayr (2015). Alternative 3'UTRs act as scaffolds to regulate membrane protein localization. Nature 522(7556): 363-367. 4 Lee, S. H. and C. Mayr (2019). "Gain of Additional BIRC3 Protein Functions through 3'-UTR-Mediated Protein Complex Formation. Mol Cell 74(4): 701-712 e709. 5 Mitschka, S. and C. Mayr (2020). Endogenous p53 expression in human and mouse is not regulated by its 3'UTR. bioRxiv: 2020.2011.2023.394197.
Title: Northern Blot Protocol v1
Description:
Northern blot is a hybridization technique to visualize and quantify RNAs from cells or tissues using a radio-labeled probe.
mRNAs only constitute a small fraction of total RNA in cells.
Through enrichment of poly(A)-tailed mRNAs, this protocol provides increased sensitivity for the detection of lowly expressed mRNAs.
In addition, RNA denaturation is achieved using glyoxal (in contrast to formaldehyde-based methods), resulting in sharper bands.
We have used this protocol extensively to detect and quantify the expression of different 3'UTR isoforms originating from the same gene 1-5.
1 Mayr, C.
and D.
P.
Bartel (2009).
Widespread shortening of 3'UTRs by alternative cleavage and polyadenylation activates oncogenes in cancer cells.
Cell 138(4): 673-684.
2 Lianoglou, S.
, V.
Garg, J.
L.
Yang, C.
S.
Leslie and C.
Mayr (2013).
Ubiquitously transcribed genes use alternative polyadenylation to achieve tissue-specific expression.
Genes Dev 27(21): 2380-2396.
3 Berkovits, B.
D.
and C.
Mayr (2015).
Alternative 3'UTRs act as scaffolds to regulate membrane protein localization.
Nature 522(7556): 363-367.
4 Lee, S.
H.
and C.
Mayr (2019).
"Gain of Additional BIRC3 Protein Functions through 3'-UTR-Mediated Protein Complex Formation.
Mol Cell 74(4): 701-712 e709.
5 Mitschka, S.
and C.
Mayr (2020).
Endogenous p53 expression in human and mouse is not regulated by its 3'UTR.
bioRxiv: 2020.
2011.
2023.
394197.
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