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Protocol for immunodot blot detection of UVB photoproducts in extracellular DNA.

ABSTRACT: UV radiation induces the formation of adducts in genomic DNA within cells that are later found to be present in cell-free fractions associated with extracellular vesicles (EVs) outside of cells. Here, we present a protocol for isolating UV photoproducts in extracellular DNA released from UVB-irradiated cells via differential centrifugation. We then detail steps for monitoring the DNA adducts using DNA immunoblotting. This protocol can be applied for detection of DNA adducts in EVs from cell culture and skin explant models. For complete details on the use and execution of this protocol, please refer to Carpenter et al.¹.

SUBMITTER: Carpenter MA

PROVIDER: S-EPMC10835299 | biostudies-literature | 2024 Jan

REPOSITORIES: biostudies-literature

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Protocol for immunodot blot detection of UVB photoproducts in extracellular DNA.

Carpenter M Alexandra MA Yerrapragada Sri S Alex Aleena A Kemp Michael G MG

STAR protocols 20240119 1

UV radiation induces the formation of adducts in genomic DNA within cells that are later found to be present in cell-free fractions associated with extracellular vesicles (EVs) outside of cells. Here, we present a protocol for isolating UV photoproducts in extracellular DNA released from UVB-irradiated cells via differential centrifugation. We then detail steps for monitoring the DNA adducts using DNA immunoblotting. This protocol can be applied for detection of DNA adducts in EVs from cell cult ...[more]

PMID: 38244199

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Project description:BackgroundNorthern blotting is still used as a gold standard for validation of the data obtained from high-throughput whole transcriptome-based methods. However, its disadvantages of lower sensitivity, labor-intensive operation, and higher quality of RNA required limit its utilization in a routine molecular biology laboratory to monitor gene expression at RNA level. Therefore, it is necessary to optimize the traditional Northern protocol to make the technique more applicable for standard use.ResultsIn this paper, we report modifications and tips used to improve the traditional Northern protocol for the detection of mRNAs in total RNA. To maximize the retention of specifically bound radiolabeled probes on the blot, posthybridization washes were performed under only with moderate-stringency until the level of radioactivity retained on the filter decreased to 20~50 counts per second, rather than normally under high and low stringency sequentially for scheduled time or under only high stringent condition. Successful detection of the low-expression gene using heterologous DNA probes in 20 µg of total RNA after a two-day exposure suggested an improvement in detection sensitivity. Quantitatively controlled posthybridization washes combined with an ethidium bromide-prestaining RNA procedure to directly visualize prestained RNA bands at any time during electrophoresis or immediately after electrophoresis, which made the progress of the Northern procedure to be monitored and evaluated step by step, thereby making the experiment reliable and controllable. We also report tips used in the modified Northern protocol, including the moderate concentration of formaldehyde in the gel, the accessory capillary setup, and the staining jar placed into an enamel square tray with a lid used for hybridization. Using our modified Northern protocol, eight rounds of rehybridization could be performed on a single blot. The modification made and tips used ensured the efficient proceeding of the experiment and the resulting good performance, but without using special reagents or equipment.ConclusionsThe modified Northern protocol improved detection sensitivity and made the experiment easy, less expensive, reliable, and controllable, and can be employed in a routine molecular biology laboratory to detect low-expressed mRNAs with heterologous DNA probes in total RNA.

Dataset Information

Protocol for immunodot blot detection of UVB photoproducts in extracellular DNA.

Publications

Protocol for immunodot blot detection of UVB photoproducts in extracellular DNA.

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