Evaluation of fluorescence-based methods for total vs. amplifiable DNA quantification in plasma of lung cancer patients.
In the last decade numerous reports demonstrated that free-circulating DNA in plasma/serum samples might be a promising biomarker in a number of pathologies, including cancer. Thus, choosing the reliable and efficient method of plasma DNA quantification would be an essential step prior to any clinical evaluation of cell-free DNA measurement in cancer patients. The aim of present study was to compare two highly-sensitive DNA quantification methods in regard to their applicability and effectiveness in monitoring the cell-free DNA level in the blood of patients with resectable non-small cell lung cancer. Plasma samples collected from 10 patients before any treatment, after neoadjuvant therapy and subsequent surgery, were used for DNA quantification by direct fluorescent PicoGreen staining and by real-time qPCR in SYBR Green and TaqMan probe approach using beta-actin gene as the amplifying target. The PicoGreen method demonstrated a high level of correlation with both the SYBR Green (r=0.87, P<0.0001) and TaqMan probe approach (r=0.94, P<0.0001). The total DNA content, determined by PicoGreen, proved to be several-fold higher than the amplifiable DNA amount measured by real-time qPCR. Consequently, intercalating fluorochromes, like PicoGreen, might serve as a rapid, accurate, and inexpensive alternative to real-time qPCR for routine dsDNA quantification and multicenter standardization.
Szpechcinski
A
A
Laboratory of Molecular Diagnostics, National Institute of Tuberculosis and Lung Diseases, Warsaw, Poland. a.szpechcinski@igichp.edu.pl
Struniawska
R
R
Zaleska
J
J
Chabowski
M
M
Orlowski
T
T
Roszkowski
K
K
Chorostowska-Wynimko
J
J
D002289:Carcinoma, Non-Small-Cell Lung / Q000097:blood*; D004273:DNA, Neoplasm / Q000097:blood*; D005456:Fluorescent Dyes; D006801:Humans; D008175:Lung Neoplasms / Q000097:blood*; D009930:Organic Chemicals; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
D004273:DNA, Neoplasm; D005456:Fluorescent Dyes; D009930:Organic Chemicals; C099476:PicoGreen