title: Correction for concentration overestimation of nucleic acids with phenol.
abstract: We report a computational method based on ultraviolet (UV) spectra for correcting the overestimated concentrations of nucleic acid samples contaminated with TRIzol/phenol. The derived correction formulas were validated using RNA solutions, double-stranded DNA solutions, and single-stranded oligonucleotide solutions. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) with SYBR Green was performed to assess the level of TRIzol contamination that can be tolerated for gene expression quantification. After the correction, the accuracy of the RNA concentrations was greatly improved and there was no significant difference in the threshold cycle (Ct) values for GAPDH and ACAN genes in RT-qPCR obtained for RNA contaminated with up to 0.1% TRIzol (phenol level index [PLI]∼5.8-5.9). Similarly, accuracy improvements were also observed for DNA or oligonucleotides contaminated with phenol using different concentration correction formulas. In addition, the Ct values and amplification efficiency of DNA in qPCR were not affected by TRIzol contamination below 1%. This computational method is easy and convenient to use and reduces the concentration overestimations greatly.
authors.affiliation: Department of Orthopaedics and Traumatology, The University of Hong Kong, Hong Kong. Electronic address: julee@connect.hku.hk.
authors.forename: Juliana T Y
authors.initials: JT
authors.lastname: Lee
authors.affiliation: Department of Orthopaedics and Traumatology, The University of Hong Kong, Hong Kong. Electronic address: cheungmc@hku.hk.
authors.forename: Kenneth M C
authors.initials: KM
authors.lastname: Cheung
authors.affiliation: Department of Orthopaedics and Traumatology, The University of Hong Kong, Hong Kong. Electronic address: vicleung@hku.hk.
authors.forename: Victor Y L
authors.initials: VY
authors.lastname: Leung
mesh_terms: D000818:Animals; D002417:Cattle; D004247:DNA / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification; D006146:Guanidines / Q000737:chemistry*; D006801:Humans; D051379:Mice; D010636:Phenols / Q000737:chemistry*; D012313:RNA / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification; D013056:Spectrophotometry, Ultraviolet / Q000379:methods
chemical_list: D006146:Guanidines; D010636:Phenols; C411644:trizol; D012313:RNA; D004247:DNA
keywords: DNA; NanoDrop; Oligonucleotides; Phenol; RNA; TRIzol