Four Decades of the Comet Assay: pH Optimum of Lysis Buffer Still Needs to be Elucidated

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F00216208%3A11120%2F23%3A43925916" target="_blank" >RIV/00216208:11120/23:43925916 - isvavai.cz</a>

Výsledek na webu

<a href="https://doi.org/10.2174/1871520623666230726114903" target="_blank" >https://doi.org/10.2174/1871520623666230726114903</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.2174/1871520623666230726114903" target="_blank" >10.2174/1871520623666230726114903</a>

Jazyk výsledku

angličtina

Název v původním jazyce

Four Decades of the Comet Assay: pH Optimum of Lysis Buffer Still Needs to be Elucidated

Popis výsledku v původním jazyce

The proper course and reproducibility of diagnostic techniques depend on narrowly defined reaction conditions, including the reaction pH. Nevertheless, numerous assays are affected by an inaccurately defined reaction pH. Buffers are sometimes suggested for use outside their useful pH ranges, which complicates the reproducibility of results because the buffering capacity is insufficient to retain the disclosed pH. Here, we focus on the comet assay lysis buffer, the broadly used method for quantifying DNA breaks in eukaryotic cells. The most widespread comet assay protocols employ lysis of the cells before electrophoresis in a buffer containing Triton X-100, a high concentration of NaCl, sodium sarcosinate, EDTA, and Tris, with some modifications. However, nearly all researchers report that they use Tris buffer at pH 10, and some report the pH of the Tris additive alone. Alternatively, others report the pH of the final lysis buffer. However, the lysis solution used in the comet assay is buffered at a pH outside the useful range of Tris. Tris-based buffers have a useful pH range of 7.0 - 9.0. The buffer composed of 10 mM Tris has pKa 8.10 at 25oC and 8.69 at 4oC. The cell lysis conditions used in nearly all modifications of comet assay protocols remain imprecise and uncritically employed. Despite the pH of the lysis buffer likely has negligible effect on the detection of DNA breaks, precise lysis conditions are highly important for the use of comet assay in the detection of base modifications, which are often unstable and sensitive to pH.

Název v anglickém jazyce

Four Decades of the Comet Assay: pH Optimum of Lysis Buffer Still Needs to be Elucidated

Popis výsledku anglicky

The proper course and reproducibility of diagnostic techniques depend on narrowly defined reaction conditions, including the reaction pH. Nevertheless, numerous assays are affected by an inaccurately defined reaction pH. Buffers are sometimes suggested for use outside their useful pH ranges, which complicates the reproducibility of results because the buffering capacity is insufficient to retain the disclosed pH. Here, we focus on the comet assay lysis buffer, the broadly used method for quantifying DNA breaks in eukaryotic cells. The most widespread comet assay protocols employ lysis of the cells before electrophoresis in a buffer containing Triton X-100, a high concentration of NaCl, sodium sarcosinate, EDTA, and Tris, with some modifications. However, nearly all researchers report that they use Tris buffer at pH 10, and some report the pH of the Tris additive alone. Alternatively, others report the pH of the final lysis buffer. However, the lysis solution used in the comet assay is buffered at a pH outside the useful range of Tris. Tris-based buffers have a useful pH range of 7.0 - 9.0. The buffer composed of 10 mM Tris has pKa 8.10 at 25oC and 8.69 at 4oC. The cell lysis conditions used in nearly all modifications of comet assay protocols remain imprecise and uncritically employed. Despite the pH of the lysis buffer likely has negligible effect on the detection of DNA breaks, precise lysis conditions are highly important for the use of comet assay in the detection of base modifications, which are often unstable and sensitive to pH.

Druh

J_imp - Článek v periodiku v databázi Web of Science

CEP obor

—

OECD FORD obor

30107 - Medicinal chemistry

Projekt

—

Návaznosti

V - Vyzkumna aktivita podporovana z jinych verejnych zdroju

Rok uplatnění

2023

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Název periodika

Anti-Cancer Agents in Medicinal Chemistry

ISSN

1871-5206

e-ISSN

1875-5992

Svazek periodika

23

Číslo periodika v rámci svazku

17

Stát vydavatele periodika

AE - Spojené arabské emiráty

Počet stran výsledku

6

Strana od-do

1910-1915

Kód UT WoS článku

001090859100002

EID výsledku v databázi Scopus

2-s2.0-85174080443