Similarly, 297 of the thenthereby 338 (87%) probeset targets hypothesized to be under-expressed in neoplastic tissues were also under-expressed in the validation experiments (P��0.05, MHT); in neoplastic specimens, 247 (73%) of these genes exhibited half or less of the expression seen in normal control tissues. Validation results by phenotype contrast are shown in Table 2. A list of validated up- and down-regulated probesets is shown in Tables S6 and S7, respectively. Table 2 Summary of microarray discovery and validation studies. Quantitative PCR assay for measuring RNA biomarker levels in tissue or plasma One of the most differentially up-regulated probesets in both the discovery (Fig 3A) and validation (Fig 3B) detected transcripts from KIAA1199, a gene of unknown function.
In the validation data set, probeset 1008852-HuGene_st (KIAA1199) was expressed more than 25-fold higher in colorectal neoplasia relative to non-neoplastic controls (Table S6). These results confirmed earlier reports, which found that KIAA1199 mRNA may be a candidate biomarker for colorectal adenoma . Based on our repeated observation of differential expression in neoplastic tissues, the prior evidence of up-regulated expression in both adenomas and cancers and the interesting fact that KIAA1199 has not been previously characterized in terms of structure or function, we chose KIAA1199 to test the idea that tissue expression patterns can be reflected in blood. To further explore the biomarker potential of KIAA1199 we designed a real-time PCR assay for detection of RNA transcripts derived from this locus.
Figure 3 KIAA1199 expression in colon tissue specimens. First, a SYBR-green based real-time PCR for KIAA1199 was used to confirm the tissue validation microarray data; the results were in good agreement with the microarray data for this gene (Fig 3C). Next, KIAA1199 (and GAPDH control) transcript levels were measured in RNA extracted from the plasma fraction of 40 patients with colorectal neoplasia (adenoma or adenocarcinoma) and 20 healthy controls (all categories having been confirmed by clinical pathological findings) using commercially available TaqMan qPCR assays. GAPDH RNA transcripts were detectable in all 60 plasma samples tested (Fig 4A) and moderately higher GAPDH RNA levels were observed in plasma specimens from patients diagnosed with colorectal adenomas or cancer compared to healthy donors (not significant, p values >0.
05). Higher concentrations of KIAA1199 RNA transcripts were detected in plasma from patients with colorectal neoplasia than in plasma Brefeldin_A from healthy controls (Fig 4B). KIAA1199 RNA was detected in plasma from 31 out of the 40 (77.5%) patients with colorectal neoplasia and in 6 out of the 20 (30%) neoplasia-free patients. Figure 4 Measurement of RNA levels in plasma specimens.