Detection of phenol contamination in RNA samples and its impact on qRT-PCR results

Conny Unger; Nicole Lokmer; Daniel Lehmann; Ilka M Axmann

doi:10.1016/j.ab.2019.02.002

Detection of phenol contamination in RNA samples and its impact on qRT-PCR results

Anal Biochem. 2019 Apr 15:571:49-52. doi: 10.1016/j.ab.2019.02.002. Epub 2019 Feb 8.

Authors

Conny Unger¹, Nicole Lokmer², Daniel Lehmann², Ilka M Axmann³

Affiliations

¹ QIAGEN GmbH, Qiagen Strasse 1, 40724, Hilden, Germany; Institute for Synthetic Microbiology, Heinrich Heine University Düsseldorf, Universitätsstraße 1, Düsseldorf, 40225, Germany. Electronic address: connyunger@yahoo.de.
² QIAGEN GmbH, Qiagen Strasse 1, 40724, Hilden, Germany.
³ Institute for Synthetic Microbiology, Heinrich Heine University Düsseldorf, Universitätsstraße 1, Düsseldorf, 40225, Germany.

PMID: 30742799
DOI: 10.1016/j.ab.2019.02.002

Abstract

Residual phenol, carried over from RNA purification, can alter RNA concentration measurements and is assumed to inhibit PCR. Here, we demonstrate that Impurities A260 values of spectral content profiling (SCP) UV/Vis measurements correlated with phenol concentration, whereas absorbance ratios of classical UV/Vis systems failed to reliably detect phenol in RNA samples. Phenol contamination led to over- or underestimation of RNA concentration on UV/Vis systems, whereas it had no influence on fluorometry quantification. Wrong RNA concentration results led to altered template input in qRT-PCR and consequently caused quantification cycle (Cq) shifts, although ≤ 0.01% phenol had no direct influence.

Keywords: A(260)/A(230); A(260)/A(280); Nanodrop; Nucleic acid quantification; QIAxpert; Qubit.

MeSH terms

Phenol / analysis*
RNA / chemistry*
RNA / genetics*
RNA / isolation & purification
Reverse Transcriptase Polymerase Chain Reaction*

Substances

Phenol
RNA