Study approval was obtained from independent ethics committees at Cancer Center of Sun Yat-Sen University. quality control The study was undertaken in accordance with the ethical standards of the World Medical Association Declaration of Helsinki. Pre-processing of blood samples Blood samples were collected in EDTA-containing tubes. Sample processing was performed within 2 hours after blood withdrawal. Blood was transferred into a 30-mL falcon tube and centrifuged at 1,800 rpm at room temperature for 20 minutes. Serum was removed, and cells were resuspended in 5 mL saline and 0.3 mL RNA later solution. After mixing well, the blood cell mixture was kept overnight at 4��C and stored at -80��C until used. RNA extraction and cDNA synthesis Total RNA of peripheral blood samples was extracted using RNAprep Cell Kit (Tiangen, Beijing, China) following the protocol provided by the manufacturer.

RNA integrity was checked by electrophoresis and quantified by absorption at 260 and 280 nm using a UV-visible spectrophotometer (Beckman Coulter Du? 800, Fullerton, CA). For reverse transcription, 1 ��g of total RNA, 1 ��L Oligo(dT)15 and 1 ��L dNTP were diluted in 10 ��L RNase-free water, incubated 10 minutes at 37��C and 1 ��L of 25 mmol/L EDTA was added. An 11 ��L aliquot of reaction mixture was incubated for 10 minutes at 65��C and quickly chilled on ice for 2 minutes. cDNA was stored at -80��C until used. cDNA synthesis was performed using the TIANScript M-MLV method (Tiangen Biotech, Beijing, China). Cell lines To prepare for CEA-specific RT-PCR, two cell lines, SW-480 (colon cancer cell line) and SC-7901 (gastric cancer cell line) were used.

Lymphocytes were collected from healthy volunteers without epithelial malignancy. After lymphocytes were isolated from peripheral blood by gradient centrifugation, the mononuclear cell layer was collected. Cell lines were serially diluted (10-fold) in 2 �� 107 to 5 �� 107 lymphocytes to give carcinoma cell: lymphocyte ratios ranging from 1:10 to 1:107. CEA mRNA Analysis by Real-Time Quantitative PCR Quantitative PCR was performed using the Sequence Detector System, ABI PRISM 7500 (Applied Biosystems 7500 Fast Real-Time PCR System). PCR primers and the TaqMan probes were designed using the Primer Express 1.0 software program. In this assay, the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control to normalize variations in integrity and total amount of RNA extracted.

The real-time PCR assays for GAPDH and CEA were done in separate tubes. CEA mRNA values were adjusted against GAPDH mRNA values, and the relative CEA mRNA scores were presented as (CEA mRNA/GAPDH mRNA) �� 106 for each sample. 5 ��L of the sample cDNA was used for real-time PCR in a 20 ��L reaction mixture consisting of 10 pmol appropriate primers (Invitrogen Cooperation, Brefeldin_A Japan) and 5 pmol TaqMan probe (Invitrogen Cooperation, Japan).