Journal: BioMed Research International

Article Title: Optimization of Preanalytical Variables for cfDNA Processing and Detection of ctDNA in Archival Plasma Samples

doi: 10.1155/2021/5585148

Figure Lengend Snippet: Workflow and experimental design for evaluating cfDNA yields affected by blood collection tubes (BCTs), storage times, and centrifugation regimes. RT: room temperature. Fresh purified samples are framed by green color while samples stored for 1 h, 4 h, and 24 h before plasma separation and freezing are framed with red color. ∗ The storage at RT refers to EDTA BCTs, which were at 4°C for 24 h before plasma separation. Each step is performed in technical duplicates obtained from the same BCT for corresponding storage time and centrifugation regime.

Article Snippet: To examine the impact of purification kits on the yield of cfDNA, three DNA purification kits were compared: DNeasy Blood & Tissue Kit (Qiagen), QIAamp Circulating Nucleic Acid Kit (Qiagen), and Quick-cfDNA Serum & Plasma Kit (Zymo Research). cfDNA was purified from blood samples collected from healthy volunteers ( n = 3).

Techniques: Centrifugation, Purification

Journal: BioMed Research International

Article Title: Optimization of Preanalytical Variables for cfDNA Processing and Detection of ctDNA in Archival Plasma Samples

doi: 10.1155/2021/5585148

Figure Lengend Snippet: Comparison of cfDNA purification kits. (a) The mean of cfDNA yield of duplicate purification for each volunteer and each kit with a standard deviation. (b) The mean yield of cfDNA from all three volunteers with standard deviation. P value obtained by one-way ANOVA test. Bars represent the total yield of cfDNA purified from 1 ml of plasma in technical duplicates.

Article Snippet: To examine the impact of purification kits on the yield of cfDNA, three DNA purification kits were compared: DNeasy Blood & Tissue Kit (Qiagen), QIAamp Circulating Nucleic Acid Kit (Qiagen), and Quick-cfDNA Serum & Plasma Kit (Zymo Research). cfDNA was purified from blood samples collected from healthy volunteers ( n = 3).

Techniques: Purification, Standard Deviation

Journal: Molecular Diagnosis & Therapy

Article Title: Personalized Cancer Monitoring Assay for the Detection of ctDNA in Patients with Solid Tumors

doi: 10.1007/s40291-023-00670-1

Figure Lengend Snippet: Workflow for the Invitae Personalized Cancer Monitoring assay. This tumor-informed assay utilizes whole exome sequencing of matched tumor and germline specimens to identify tumor-specific variants. A proprietary algorithm selects tumor-specific variants to design a patient-specific panel. The designed panel is then used to generate next-generation sequencing libraries, featuring anchored multiplex polymerase chain reaction chemistry, and unique molecular identifiers, from cell-free DNA (cfDNA) extracted from the patient’s plasma specimen. From the sequencing data, stringent data quality-control measures and a proprietary circulating tumor DNA (ctDNA)-calling algorithm are used to assess the ctDNA status in the patient’s specimen. FFPE formalin-fixed paraffin-embedded

Article Snippet: Cell-free DNA was then isolated from the plasma using the Maxwell RSC Circulating Cell-Free DNA Purification Kit (Promega) according to the manufacturer’s instructions.

Techniques: Sequencing, Next-Generation Sequencing, Multiplex Assay, Polymerase Chain Reaction, Formalin-fixed Paraffin-Embedded

Journal: Molecular Diagnosis & Therapy

Article Title: Personalized Cancer Monitoring Assay for the Detection of ctDNA in Patients with Solid Tumors

doi: 10.1007/s40291-023-00670-1

Figure Lengend Snippet: Assay sensitivity with different panel sizes. Patient-specific panels with 16–50 variants were assessed along with cell-free DNA input amounts (10–60 ng) and analytical thresholds (baseline and monitoring). A . The y -axis shows sensitivity (%) and the x -axis shows variant allele frequency (AF) [%]. Larger panel size, higher input amount, and baseline threshold correlated with detection at lower AFs. B . Heat plot of sensitivity data points

Article Snippet: Cell-free DNA was then isolated from the plasma using the Maxwell RSC Circulating Cell-Free DNA Purification Kit (Promega) according to the manufacturer’s instructions.

Techniques: Variant Assay

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

Journal: Thoracic Cancer

Article Title: Prognostic and predictive impact of molecular tumor burden index in non‐small cell lung cancer patients

doi: 10.1111/1759-7714.15098

Figure Lengend Snippet: ctDNA level of pretreatment samples. (a) Kaplan–Meier survival analysis of overall survival (OS) stratified by ctDNA status of baseline. (b) Kaplan–Meier survival analysis of progression‐free survival (PFS) stratified by ctDNA status of baseline. (c) Sum of longest axial diameters (SLD) in patients with ctDNA‐positive versus those with ctDNA‐negative. (d) Correlation between baseline molecular tumor burden index (mTBI) and variant allele frequency (VAF). ctDNA, circulating tumor DNA.

Article Snippet: Circulating cell‐free DNA was isolated from plasma using the QIAsymphony circulating DNA kit (Qiagen).

Techniques: Variant Assay