Patients provided a blood sample prior to endoscopy, and anonymous clinical data was collected from each subject. Informed consent was obtained as approved by the institutions’ Research Ethics Board and Joint Ethics Committee. All subjects were 21 years or older, and subjects with known, blood-borne infectious diseases (e.g. HIV, HCV) were excluded. Isolation of Whole Blood RNA All blood specimens were collected prior to colonoscopy using PAXgene™tubes (PreAnalytix) and processed according to the PAXgene Blood
RNA Kit protocol. Blood specimens for RNA isolation and downstream testing were kept refrigerated after collection and during transportation to GeneNews (Malaysia) Laboratory, a Standards Malaysia ISO-17025 accredited laboratory at Mount Miriam Cancer Hospital in Penang. find more RNA quality was assessed Selleck P505-15 using Agilent 2100 Bioanalyzer RNA 6000 Nano Kit (Agilent Technologies). RNA quantity was determined by QNZ mw absorbance at 260 nm in a DU800 Spectrophotometer (Beckman-Coulter). The acceptance criteria for the RNA samples are: RIN
≥ 7.0; rRNA ratio ≥ 1.0 and a validated Agilent Bioanalyzer scan. Quantitative Reverse-Transcriptase Polymerase Chain Reaction Quantitative reverse-transcriptase real-time RT-PCR reaction procedures for the seven gene biomarkers (ANXA3, CLEC4D, TNFAIP6, LMNB1, PRRG4, VNN1 and the duplex partner or reference gene, IL2RB) have been described previously . Briefly, one microgram of RNA was reverse-transcribed into single-stranded complementary DNA (cDNA) using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) in 1X RT reaction. For qPCR, 20 ng cDNA was mixed with QuantiTect® Probe PCR Master Mix (Qiagen) 2-hydroxyphytanoyl-CoA lyase and Taqman® dual-labeled probe and primers corresponding to the gene-of-interest and reference
gene, IL2RB, in a 25 μL reaction volume. PCR amplification was performed using a 7500 Real-Time PCR System (Applied Biosystems). Up to 4 samples – each sample run in duplicate – can be analyzed on a single plate. Water was added to the outer wells to ensure proper temperature equilibrium. No-template controls (NTC) containing water and master mix were added to column 12 to check for possible reagent or test contamination. Column 2 and column 11 were designated for pooled blood RNA (PBR) samples for monitoring the performance of both RT and qPCR steps. PBR was prepared from blood RNA isolated from specimens collected from volunteers. Wells from row 2 to row 7 were designated for the corresponding six biomarkers, ANXA3, CLEC4D, TNFAIP6, LMNB1, PRRG4 and VNN1. IL2RB served as the reference gene for the six biomarkers. Results Over the two-year period 2007 to 2009, we collected 421 blood samples, of which about one quarter were obtained from CRC patients. More than 95% of the samples passed quality control criteria (Table 1).