Feb 02, 2025
5-ethynyl uridine labeling of nascent mitochondrial genome transcription
- Tejashree Waingankar1,
- Samantha C Lewis1
- 1UC Berkeley
Protocol Citation: Tejashree Waingankar, Samantha C Lewis 2025. 5-ethynyl uridine labeling of nascent mitochondrial genome transcription. protocols.io https://dx.doi.org/10.17504/protocols.io.261gero6wl47/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working as of 1/1/2025.
Created: November 03, 2024
Last Modified: February 02, 2025
Protocol Integer ID: 111778
Keywords: Fluorescence microscopy, Transcription, mitochondria, mtDNA, click chemistry
Funders Acknowledgements:
Sloan Research Foundation
Grant ID: FG-2023-20384
NSF
Grant ID: 2339182
Abstract
This protocol details a method for 5-ethynyl uridine labeling of nascent mitochondrial genome transcription coupled with immunofluorescence detection of mtDNA-binding proteins in cultured cells. The result is a fluorescent readout of localized mitochondrial gene transcription compatible with high-resolution microscopy.
Guidelines
This protocol was developed to work with cultured mammalian cell lines. It also works with cultured mammalian neurons.
Materials
Buffers and reagents:
- 1 mM Triptolide diluted in DMSO
- 0.5 M 5-ethynyl uridine (EU) in DMSO
- Mitotracker Deep Red (MTDR) MitoTracker™ Deep Red FM - Special PackagingThermo FisherCatalog #M22426
- Click-iT™ Plus EdU Alexa Fluor™ 647 Imaging KitThermo FisherCatalog #C10640
- Permeabilization solution: DPBS + 0.1% Triton X-100
- Blocking buffer: 0.1% TBST + 1% BSA
- culture media: Complete MEM: For 50 ml
| A | B | |
| Horse serum | 5 ml | |
| Pen/strep antibiotic | 500 µl | |
| L-Glutamine | 500 µl | |
| MEM vitamin supplement | 500 µl | |
| MEM | 43.5 ml |
Method
Method
1d 14h 35m
1d 14h 35m
Culture cells in vitro for 24:00:00 in 300 µL complete MEM on a coverslip or in a 35 mm glass bottom culture dish.
1d
Remove the 300 µL of culture media and add pre-warmed 1 mL of complete culture media.
Add 1 µL of 1 millimolar (mM) Triptolide to the media and incubate at 37 °C for 01:00:00 .
1h
Add 1 µL of 0.5 Molarity (M) EU to the media and incubate for 03:00:00 at 37 °C .
3h
After 2h, add 50 nanomolar (nM) MTDR to the culture media.
Remove the media and replace it with fresh, complete culture media. Incubate at 37 °C for 00:10:00 .
10m
Fix with pre-warmed 4% PFA for 00:20:00 at Room temperature .
20m
Add 200 µL of 1 Molarity (M) Glycine to quench the reaction for 00:05:00 at Room temperature .
5m
Add 2 mL of permeabilization solution for 00:10:00 at Room temperature .
10m
Replace the permeabilization solution with the blocking buffer for 00:10:00 at Room temperature .
10m
Replace the blocking buffer solution with primary antibody solution diluted in blocking buffer; incubate at 4 °C Overnight .
8h
Prepare a click reaction buffer mixture as described in the manufacturer's protocol.
Incubate at Room temperature for 00:30:00 while protected from light.
30m
Remove the solution completely and wash gently with 1 mL of blocking buffer.
Replace with freshly prepared secondary antibody solution diluted in blocking buffer; incubate for 01:00:00 at Room temperature , protected from light.
1h
Wash once with the blocking buffer for 00:10:00 .
10m
Cover cells in 500 µL DPBS and proceed with fluorescence microscopy imaging.
Protocol references
Jao CY, Salic A. Exploring RNA transcription and turnover in vivo by using click chemistry. Proc Natl Acad Sci U S A. 2008 Oct 14;105(41):15779-84. doi: 10.1073/pnas.0808480105. Epub 2008 Oct 7. PMID: 18840688; PMCID: PMC2572917.
Fantoni NZ, El-Sagheer AH, Brown T. A Hitchhiker's Guide to Click-Chemistry with Nucleic Acids. Chem Rev. 2021 Jun 23;121(12):7122-7154. doi: 10.1021/acs.chemrev.0c00928. Epub 2021 Jan 14. PMID: 33443411.
Acknowledgements
We thank the Lewis Lab for helpful discussion.