Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

Search BRD Papers

Comparison of 7 methods for extracting cell-free DNA from serum samples of colorectal cancer patients.

Author(s): Fong SL, Zhang JT, Lim CK, Eu KW, Liu Y

Publication: Clin Chem, 2009, Vol. 55, Page 587-9

PubMed ID: 19246404 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared the DNA yield from serum when extraction was with seven different methods and quantified using fluorometry, real-time PCR, and real-time PCR after bisulfite conversion. The size of isolated DNA was also compared by electrophoresis. Twelve pooled serum specimens were obtained from the serum of 67 patients with colorectal carcinoma (details of blood procurement and processing not provided). Serum is assumed to have been frozen prior to use. DNA was extracted from serum using a phenol/chloroform method with addition of glycogen, an in-house sodium iodide method, an in-house guanidine-resin based method, the QIAamp DNA Blood Midi Kit with carrier RNA, the ChargeSwitch 1-mL Serum Kit, the Zymo Research Serum DNA Kit, or the Puregene DNA Purification System Cell and Tissue Kit. DNA was quantified by Quant-iT dsDNA HS assay (Invitrogen) and by real-time PCR amplification of the epithelial cadherin 1(CDH1) gene. DNA was also bisulfite-converted and quantified by amplification of a region of ACTB without CpG sites. The size distribution of DNA was investigated by electrophoresis of DNA isolated from 1 mL of serum.

   Summary of Findings:

   The DNA concentration as measured by fluorometry and amplification of CDH1 was highest when extraction was with the phenol/chloroform method with addition of glycogen or an in-house sodium iodide-based method followed by the QIAamp DNA Blood Midi Kit with carrier RNA. Importantly, these three methods were the only methods that yielded sufficient DNA to amplify ACTB after bisulfite conversion. Electrophoresis revealed visible bands of 200, 400, and 500 bp when extraction was with the phenol/chloroform based method with addition of glycogen or the in-house sodium iodide-based method but only the 500 bp band was visible when extraction was with the other methods. The authors state the loss of 200 and 400 bp bands indicated poor recovery of nucleosomal DNA. While not significantly different from the phenol-chloroform based method, the sodium iodide-based method yielded more amplifiable DNA overall and at a lower cost after bisulfite conversion, making it the authors method of choice.

    

   Biospecimens

   • Bodily Fluid - Serum

   Preservative Types

   • Frozen

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   DNA	Fluorometry
   DNA	Electrophoresis
   DNA	Real-time qPCR
   DNA	Bisulfite conversion assay

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Analyte isolation method	Phenol/chloroform method with addition of glycogen QIAamp DNA Blood Midi Kit with carrier RNA ChargeSwitch 1-mL Serum Kit Zymo Research Serum DNA Kit Puregene DNA Purification System Cell and Tissue Kit In-house sodium iodide method In-house guanidine-resin based method
   Real-time qPCR Specific	Targeted nucleic acid	CDH1
   Bisulfite conversion assay Specific	Targeted nucleic acid	ACTB
   Real-time qPCR Specific	Technology platform	Amplification of bisulfite converted DNA Fluorometry Standard real-time PCR

   View more