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Correction for concentration overestimation of nucleic acids with phenol
Juliana T Y Lee1, Kenneth M C Cheung1, Victor Y L Leung1
1Department of Orthopaedics and Traumatology, The University of Hong Kong, Hong Kong.
Analytical Biochemistry
|August 19, 2014
Summary
A new computational method using UV spectra accurately corrects overestimated nucleic acid concentrations caused by TRIzol/phenol contamination. This improves RNA and DNA quantification accuracy for molecular biology applications.
Area of Science:
- Molecular Biology
- Biochemistry
- Analytical Chemistry
Background:
- TRIzol and phenol contamination commonly lead to overestimated nucleic acid concentrations.
- Accurate nucleic acid quantification is crucial for downstream molecular biology applications like gene expression analysis.
Purpose of the Study:
- To develop and validate a computational method for correcting nucleic acid concentration overestimations due to TRIzol/phenol contamination.
- To assess the impact of TRIzol contamination on the accuracy of RNA and DNA quantification and subsequent molecular analyses.
Main Methods:
- A computational method utilizing ultraviolet (UV) spectra was developed to correct overestimated nucleic acid concentrations.
- Correction formulas were validated using RNA, double-stranded DNA, and single-stranded oligonucleotide solutions.
- Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and quantitative polymerase chain reaction (qPCR) were used to assess the impact of contamination on gene expression and DNA amplification.
Main Results:
- The computational method significantly improved the accuracy of RNA concentration measurements.
- RT-qPCR showed no significant difference in threshold cycle (Ct) values for target genes when RNA was contaminated with up to 0.1% TRIzol after correction.
- Similar accuracy improvements were observed for DNA and oligonucleotide quantification.
- qPCR analysis indicated that TRIzol contamination below 1% did not affect DNA Ct values or amplification efficiency.
Conclusions:
- The developed computational method provides an easy and effective way to correct nucleic acid concentration overestimations caused by TRIzol/phenol.
- This method enhances the reliability of nucleic acid quantification, particularly for gene expression studies using RT-qPCR.
- The findings demonstrate the robustness of the correction method across different nucleic acid types and contamination levels.
