Limits...
A rapid non-radioactive technique for measurement of repair synthesis in primary human fibroblasts by incorporation of ethynyl deoxyuridine (EdU).

Limsirichaikul S, Niimi A, Fawcett H, Lehmann A, Yamashita S, Ogi T - Nucleic Acids Res. (2009)

Bottom Line: We have established a rapid and accurate procedure for measuring UDS by replacement of thymidine with 5-ethynyl-2'-deoxyuridine (EdU).We demonstrate that the EdU incorporation assay is compatible with conventional techniques such as immunofluorescent staining and labeling of cells with micro-latex beads.Importantly, we can complete the entire UDS assay within half a day from preparation of the assay coverslips; this technique may prove useful as a method for XP diagnosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Medicine, Atomic Bomb Disease Institute, Nagasaki University, Nagasaki, 852-8523 Japan.

ABSTRACT
Xeroderma pigmentosum (XP) is an autosomal recessive genetic disorder. Afflicted patients show extreme sun-sensitivity and skin cancer predisposition. XP is in most cases associated with deficient nucleotide excision repair (NER), which is the process responsible for removing photolesions from DNA. Measuring NER activity by nucleotide incorporation into repair patches, termed 'unscheduled DNA synthesis (UDS)', is one of the most commonly used assays for XP-diagnosis and NER research. We have established a rapid and accurate procedure for measuring UDS by replacement of thymidine with 5-ethynyl-2'-deoxyuridine (EdU). EdU incorporated into repair patches can be directly conjugated to fluorescent azide derivatives, thereby obviating the need for either radiolabeled thymidine or denaturation and antibody detection of incorporated bromodeoxyuridine (BrdU). We demonstrate that the EdU incorporation assay is compatible with conventional techniques such as immunofluorescent staining and labeling of cells with micro-latex beads. Importantly, we can complete the entire UDS assay within half a day from preparation of the assay coverslips; this technique may prove useful as a method for XP diagnosis.

Show MeSH

Related in: MedlinePlus

EdU assay is compatible with immunostaining. (A) Normal 48BR and XPG-deficient XP20BE fibroblasts were co-cultured on coverslips, UVC irradiated (20 J/m2), followed by 2-h incubation with 10 μM EdU. Fixed cells were then immunostained with rabbit anti-XPG antibody (red), followed by conjugation of Alexa fluor 488-azide to incorporated EdU (green). (B) UV-induced EdU incorporation in quiescent cells. 48BR cells were UVC irradiated (20 J/m2), followed by 2-h incubation with 10 μM EdU. Fixed cells were then immunostained with rabbit anti-ki67 antibody (red), followed by conjugation of Alexa fluor 488-azide to incorporated EdU (green). Triangle, normal arrow and diamond arrow indicate S-phase, G0 and G1 (or G2/M), respectively. Detailed experimental conditions are described in ‘Materials and methods’ section.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2651789&req=5

Figure 3: EdU assay is compatible with immunostaining. (A) Normal 48BR and XPG-deficient XP20BE fibroblasts were co-cultured on coverslips, UVC irradiated (20 J/m2), followed by 2-h incubation with 10 μM EdU. Fixed cells were then immunostained with rabbit anti-XPG antibody (red), followed by conjugation of Alexa fluor 488-azide to incorporated EdU (green). (B) UV-induced EdU incorporation in quiescent cells. 48BR cells were UVC irradiated (20 J/m2), followed by 2-h incubation with 10 μM EdU. Fixed cells were then immunostained with rabbit anti-ki67 antibody (red), followed by conjugation of Alexa fluor 488-azide to incorporated EdU (green). Triangle, normal arrow and diamond arrow indicate S-phase, G0 and G1 (or G2/M), respectively. Detailed experimental conditions are described in ‘Materials and methods’ section.

Mentions: We anticipate applying the EdU-based UDS assay system to clinical diagnoses, in which rigorous intra-assay controls are required. We therefore assessed the compatibility of the UDS assay with other standard techniques that use internal references. We found that fluorescent-azide conjugation to EdU is fully compatible with immunofluorescent staining (Figure 3A and B).Figure 3.


A rapid non-radioactive technique for measurement of repair synthesis in primary human fibroblasts by incorporation of ethynyl deoxyuridine (EdU).

Limsirichaikul S, Niimi A, Fawcett H, Lehmann A, Yamashita S, Ogi T - Nucleic Acids Res. (2009)

EdU assay is compatible with immunostaining. (A) Normal 48BR and XPG-deficient XP20BE fibroblasts were co-cultured on coverslips, UVC irradiated (20 J/m2), followed by 2-h incubation with 10 μM EdU. Fixed cells were then immunostained with rabbit anti-XPG antibody (red), followed by conjugation of Alexa fluor 488-azide to incorporated EdU (green). (B) UV-induced EdU incorporation in quiescent cells. 48BR cells were UVC irradiated (20 J/m2), followed by 2-h incubation with 10 μM EdU. Fixed cells were then immunostained with rabbit anti-ki67 antibody (red), followed by conjugation of Alexa fluor 488-azide to incorporated EdU (green). Triangle, normal arrow and diamond arrow indicate S-phase, G0 and G1 (or G2/M), respectively. Detailed experimental conditions are described in ‘Materials and methods’ section.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2651789&req=5

Figure 3: EdU assay is compatible with immunostaining. (A) Normal 48BR and XPG-deficient XP20BE fibroblasts were co-cultured on coverslips, UVC irradiated (20 J/m2), followed by 2-h incubation with 10 μM EdU. Fixed cells were then immunostained with rabbit anti-XPG antibody (red), followed by conjugation of Alexa fluor 488-azide to incorporated EdU (green). (B) UV-induced EdU incorporation in quiescent cells. 48BR cells were UVC irradiated (20 J/m2), followed by 2-h incubation with 10 μM EdU. Fixed cells were then immunostained with rabbit anti-ki67 antibody (red), followed by conjugation of Alexa fluor 488-azide to incorporated EdU (green). Triangle, normal arrow and diamond arrow indicate S-phase, G0 and G1 (or G2/M), respectively. Detailed experimental conditions are described in ‘Materials and methods’ section.
Mentions: We anticipate applying the EdU-based UDS assay system to clinical diagnoses, in which rigorous intra-assay controls are required. We therefore assessed the compatibility of the UDS assay with other standard techniques that use internal references. We found that fluorescent-azide conjugation to EdU is fully compatible with immunofluorescent staining (Figure 3A and B).Figure 3.

Bottom Line: We have established a rapid and accurate procedure for measuring UDS by replacement of thymidine with 5-ethynyl-2'-deoxyuridine (EdU).We demonstrate that the EdU incorporation assay is compatible with conventional techniques such as immunofluorescent staining and labeling of cells with micro-latex beads.Importantly, we can complete the entire UDS assay within half a day from preparation of the assay coverslips; this technique may prove useful as a method for XP diagnosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Medicine, Atomic Bomb Disease Institute, Nagasaki University, Nagasaki, 852-8523 Japan.

ABSTRACT
Xeroderma pigmentosum (XP) is an autosomal recessive genetic disorder. Afflicted patients show extreme sun-sensitivity and skin cancer predisposition. XP is in most cases associated with deficient nucleotide excision repair (NER), which is the process responsible for removing photolesions from DNA. Measuring NER activity by nucleotide incorporation into repair patches, termed 'unscheduled DNA synthesis (UDS)', is one of the most commonly used assays for XP-diagnosis and NER research. We have established a rapid and accurate procedure for measuring UDS by replacement of thymidine with 5-ethynyl-2'-deoxyuridine (EdU). EdU incorporated into repair patches can be directly conjugated to fluorescent azide derivatives, thereby obviating the need for either radiolabeled thymidine or denaturation and antibody detection of incorporated bromodeoxyuridine (BrdU). We demonstrate that the EdU incorporation assay is compatible with conventional techniques such as immunofluorescent staining and labeling of cells with micro-latex beads. Importantly, we can complete the entire UDS assay within half a day from preparation of the assay coverslips; this technique may prove useful as a method for XP diagnosis.

Show MeSH
Related in: MedlinePlus