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Rapid removal of dextran sulfate sodium from tissue RNA preparations for measurement of inflammation biomarkers

Author:
Juritsch, Anthony F., Moreau, Régis
Source:
Analytical biochemistry 2019 v.579 pp. 18-24
ISSN:
0003-2697
Subject:
biomarkers, complementary DNA, dextran sulfate, inflammation, interleukin-8, lithium chloride, macrophages, messenger RNA, mice, quantitative polymerase chain reaction, reverse transcriptase polymerase chain reaction, reverse transcription, silica, systematic review, tissues
Abstract:
Dextran sulfate sodium (DSS) present in the tissues of DSS-treated laboratory animals inhibits quantitative real-time qPCR (RT-qPCR) and thus may be source of experimental errors. A recent systematic review concluded that the reporting of experimental method was insufficient in a majority of DSS studies and contributed to the poor reproducibility of experiments. Here we compared two DSS cleanup protocols applied to mouse tissue RNA preparations based on silica membrane spin column and lithium chloride precipitation. In absence of cleanup, exogenous DSS significantly inhibited reverse transcription and cDNA amplification at concentrations of 5 × 10−3 g/L and above during the quantification of IL8 mRNA levels in THP-1 macrophages. Silica membrane spin columns removed DSS from mouse RNA preparations and eliminated DSS-induced inhibition of qPCR. Mouse RNA isolated from DSS-treated tissues and purified with silica membrane spin columns was suitable for RT-qPCR and assessment of inflammatory biomarkers.
Agid:
6448231