Genomic Profiling of Circulating Plasma RNA for the Analysis of Cancer

Abstract

(sVCAM, sICAM, CRP), and manual methods were used on the other assayed selectins. A few measurements do not fit any discernable pattern, notably plasma sVCAM-1 on day 3 at 4 and 21°C; plasma sE-selectin on day 1 at 4°C and day 3 at all temperatures, whole blood sE-selectin at 4°C on day 1 and after freeze-thaw cycle 4; and plasma CRP on day 1 at 4°C and after freeze-thaw cycle 3. These aberrant values were likely analytic artifacts, because removal of values Ͼ4 SDs from the mean or adjustment for run-to-run variability did not change the results qualitatively. The large CRP difference observed after freeze-thaw cycle 3 but not after cycles 4 or 5 suggests assay error or possibly CRP release from LDL and complement factors. It has been demonstrated that a portion of systemic CRP is bound to cholesterol in modified LDL particles (17 ) and to different complement factors (18 ). Interaction of CRP with cholesterol and complement factors might hamper the detection of CRP in the assay used. No data were available for sE-selectin, but it is possible that some sE-selectin is bound to different circulating leukocyte types or microvesicles derived from endothelial cells. Freeze-thaw cycles might release these bound forms of sE-selectin, leading to an increase in detectable sE-selectin.Possible effects of residual platelets in the plasma on marker values obtained are not addressed in our study. sVCAM-1, sICAM-1, and sE-selectin, however, are derived mainly from endothelial cells and not from platelets. Although the presence of platelets in EDTA plasma might contribute to the absolute amounts of these markers, it does not influence their stability. In contrast, sP-selectin is mainly derived from platelets, a characteristic that may explain the variance observed in sP-selectin at different time and temperature points.Our results show that sP-selectin is unstable under all storage conditions and requires immediate assay. The other cardiovascular risk markers evaluated were stable when stored in whole blood samples for several days at room temperature. sVCAM-1, sICAM-1, and CRP were stable at 4 and 21°C in plasma or whole blood for 5 days and sE-selectin for 2 days. These findings have important implications for clinical studies measuring these markers, reducing the need for immediate transfer of samples to a laboratory for processing and analysis.

Relationship Between Tumor and Plasma Levels of hTERT mRNA in Patients with Colorectal Cancer: Implications for Monitoring of Neoplastic Disease

“…Unfortunately, data on paired tumor samples was not provided in this study; however, sensitivity and specificity of the assay for CRC detection were estimated to be 98% and 64%, respectively (24). Of note, a recent study of gene expression profiling from plasma of 12 CRC patients and 8 healthy subjects did not include hTERT in the list of genes found to be differentially up-regulated in cancer (10). Under-represented probes of the hTERT gene in the microarray likely explain the failure of this approach in detecting differential expression of this gene.…”

“…RNA can be isolated from plasma, and plasma-derived RNA can be reverse transcribed and amplified by PCR (7,9). Recently, analysis of circulating RNA has been also reported as a potential approach for gene expression profiling in cancer patients (10).…”

Presence and Characterization of Cell-Free Seminal RNA in Healthy Individuals: Implications for Noninvasive Disease Diagnosis and Gene Expression Studies of the Male Reproductive System

“…Much interest has been focused in recent years on the applications of cell-free nucleic acids in prenatal diagnosis (6,7 ), oncology (8,9 ), and the etiopathology of other pathologic disorders (10 ). Considering previous results regarding cfRNA in human plasma (11)(12)(13), saliva (14 ), and bronchial lavage fluid (15 ), and the rapid development of molecular biology techniques, we believe cell-free seminal RNA (cfsRNA) may represent a new source of noninvasive biomarkers for the male reproductive system and germline epigenetics.…”

Circulating nucleic acids as a new diagnostic tool

“…Using microarray analysis for the detection of the whole human serum transcriptome is a current promising approach. Li et al was one of the first to target the serum transcriptome in patients with head and neck squamous cell carcinoma, using high density oligonucleotide microarrays [49,88]. Recent studies have been dominated by three new detection methods: nucleic acid mass spectrometry, digital PCR and second generation DNA sequencing.…”