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Rapid removal of dextran sulfate sodium from tissue RNA preparations for measurement of inflammation biomarkers
- Source :
- Analytical Biochemistry. 579:18-24
- Publication Year :
- 2019
- Publisher :
- Elsevier BV, 2019.
-
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.
- Subjects :
- Male
THP-1 Cells
animal diseases
medicine.medical_treatment
Biophysics
digestive system
01 natural sciences
Biochemistry
Specimen Handling
Mice
03 medical and health sciences
chemistry.chemical_compound
Spin column-based nucleic acid purification
Complementary DNA
medicine
Animals
Humans
RNA, Messenger
Interleukin 8
Molecular Biology
030304 developmental biology
Inflammation
0303 health sciences
Messenger RNA
Dextran Sulfate
010401 analytical chemistry
RNA
Cell Biology
Colitis
digestive system diseases
Reverse transcriptase
0104 chemical sciences
Mice, Inbred C57BL
stomatognathic diseases
Cytokine
chemistry
Cytokines
Lithium chloride
Scientific Experimental Error
Biomarkers
Subjects
Details
- ISSN :
- 00032697
- Volume :
- 579
- Database :
- OpenAIRE
- Journal :
- Analytical Biochemistry
- Accession number :
- edsair.doi.dedup.....944cf323da5938ed94b211e7c519c17f