Journal: International Journal of Molecular Sciences

Article Title: Peptide-Affinity Precipitation of Extracellular Vesicles and Cell-Free DNA Improves Sequencing Performance for the Detection of Pathogenic Mutations in Lung Cancer Patient Plasma

doi: 10.3390/ijms21239083

Figure Lengend Snippet: Effect of plasma pre-clearing on DNA recovery and detection of EV markers in PA precipitated material. ( A ) Plasma from six NSCLC donors was either not pre-cleared, or pre-cleared at 3000× g or 17,000× g for 15 min. The post-centrifugation pellet was retained, resuspended in nuclease-free water and DNA was extracted. Peptide affinity (PA) precipitation was performed on equivalent volumes of either non-precleared or pre-cleared plasma and DNA (PA-DNA) was isolated using the Plasma/Serum Cell-Free Circulating DNA Purification Mini Kit (Norgen Biotek). Cell-free DNA (cf-DNA) was obtained from equivalent volumes of plasma using the same DNA isolation kit for comparison ( n = 6; * p < 0.05). A representative overlay of the DNA profiles of ( B ) PA-DNA or ( C ) cf-DNA from plasma (Donor #1) using either no pre-clearing or 3000× g or 17,000× g pre-clearing is shown. ( D ) A representative western blot ( n = 3) of Vn96 PA precipitated material from 1 mL of plasma from donors with benign lung disease or NSCLC is shown. Canonical EV markers CD63, CD9, HSC70, and flotillin-1 were detected using specific antibodies. A vehicle control sample (without Vn96) was included as a negative control (−). In addition, calnexin, apolipoprotein A1 (Apo-A1), and albumin, which are common co-contaminants of EV isolations from plasma, were also detected using specific antibodies. Plasma protein lysate was included as a positive control for non-EV-associated plasma proteins.

Article Snippet: DNA Isolation: DNA was isolated directly from up to 0.5 mL of plasma (cf-DNA) using the Plasma/Serum Cell-Free Circulating DNA Purification Kit (Norgen Biotek, Thorold, ON, Canada) according to the manufacturer’s instructions.

Techniques: Clinical Proteomics, Centrifugation, Isolation, DNA Purification, DNA Extraction, Comparison, Western Blot, Control, Negative Control, Positive Control