Evaluation of fluorescence-based methods for total vs. amplifiable DNA quantification in plasma of lung cancer patients.
Author(s): Szpechcinski A, Struniawska R, Zaleska J, Chabowski M, Orlowski T, Roszkowski K, Chorostowska-Wynimko J
Publication: J Physiol Pharmacol, 2008, Vol. 59 Suppl 6, Page 675-81
PubMed ID: 19218694 PubMed Review Paper? No
Purpose of Paper
The purpose of this paper was to compare DNA quantification methods and determine the effect of treatment and surgery on plasma DNA levels.
Conclusion of Paper
Mean DNA concentrations, were higher when determined using PicoGreen than those determined by TaqMan and SYBR real-time PCR. However, DNA concentrations determined by the three methods were strongly or very strongly correlated. The authors report that plasma DNA levels for 7 of the 10 patients examined differed significantly among those obtained before treatment, after neoadjuvant therapy and after surgery.
Studies
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Study Purpose
The purpose of this study was to compare DNA quantification by PicoGreen fluorometry, TaqMan real-time PCR, and SYBR real-time PCR, and to determine the effect of treatment and surgery on plasma DNA levels. Blood was obtained from 10 non-small cell lung cancer (NSCLC) patients before treatment, after neoadjuvant therapy and after surgery. Plasma was obtained by centrifugation and stored frozen at -80ºC until DNA extraction using the QIAamp DNA Blood Midi kit. DNA was stored at -20ºC until quantification with PicoGreen and by real-time PCR amplification of β-Actin using SYBR and TaqMan chemistries.
Summary of Findings:
Mean DNA concentrations were 5-fold higher when determined using PicoGreen than by TaqMan real-time PCR, and 10-fold higher than by SYBR real-time PCR (p<0.03). However, concentrations determined via the PicoGreen method were strongly correlated with those determined by SYBR green real-time PCR (r=0.87, p<0.0001) and very strongly correlated with those determined by TaqMan real-time PCR (r=0.94, p<0.0001). Further, concentrations determined by real-time PCR using SYBR and TaqMan were very strongly correlated (r=0.96, p<0.0001). The authors report that plasma DNA levels for 7 of the 10 patients examined differed significantly among those obtained before treatment, after neoadjuvant therapy and after surgery.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform DNA Real-time qPCR DNA Fluorometry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Time of biospecimen collection Before treatment
After neoadjuvant therapy
After surgery
Real-time qPCR Specific Technology platform TaqMan probes
SYBR green
PicoGreen fluoremetry
Real-time qPCR Specific Detection method TaqMan
SYBR green