All blood samples from patients diagnosed with mCRC were obtained through a study with approval by the MD Anderson Institutional Review Board, and all participants provided written informed consent (NCT01730586). Blood samples were collected at baseline from mCRC patients before clinical trial treatment. Blood samples of cancer-free individuals were obtained from outpatients of Johns Hopkins Community Physicians, with approval by the Johns Hopkins Medicine Institutional Review Board. All participants provided written informed consent. mCRC and cancer-free patient characteristics are provided in fig. S7B. All blood samples were collected in cell preparation tubes (BD) and processed within 1 hour with centrifugation (3000g) for 30 min, and then plasma was aliquoted to 1.8-ml cryovials and stored at −80°C until analysis.

All cfDNA was extracted using 2-ml NeoGeneStar Circulating Cell Free DNA kit (NeoGeneStar) according to the manufacturer’s instructions. Briefly, 2.0 ml of plasma was stabilized with pretreatment buffer and then digested in a solution containing 1× protease buffer and 100 μl of proteinase K (Invitrogen). DNA was then extracted with supplied chaotropic salts, washed by a series of magnetic decantation steps, and eluted into 20 μl of DNA elution buffer (Zymo Research). Extracted cfDNA was quantified by quantitative PCR using primers recognizing β-globin (forward primer: 5′-TGAAGGCTCATGGCAAGAAAG-3′; reverse primer: 5′-GAGGTTGTCCAGGTGAGCCA-3′). PCR performed using 10× PCR buffer (Thermo Fisher Scientific) to yield a final volume of 25 μl and a final working concentration of 3.5 mM MgCl2, 200 μM of each dNTP, and Platinum Taq polymerase (0.04 U/μl) (Thermo Fisher Scientific). Cycling conditions were 95°C for 5 min, followed by 50 cycles of 95°C for 5 s, 65°C for 30 s, and 72°C for 30 s. Standards for quantification were created by serial dilution of human male genomic DNA (Promega). The resulting DNA was bisulfite-converted using the EZ DNA Methylation-Lightning Kit (Zymo Research) according to the manufacturer’s protocol and eluted into a final volume of 12 μl. Final bisulfite-treated–cfDNA yields were quantified by β-actin PCR, as described for control DNA above.

Note: The content above has been extracted from a research article, so it may not display correctly.

Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.

We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.