Abstract
BackgroundMinimizing the effects of transportation on the properties of biological material is a major challenge for the scientific community. The viability of cells is important in cases where their study is urgent for evaluation of treatment response or for the study of cancer progression. Circulating tumor cells (CTCs) constitute a cell subpopulation with great importance for oncologists, because of their prognostic value. Detection and isolation of CTCs from blood samples is a routine activity in many laboratories, but concerns exist with regard to the maintenance of the cells during transportation. In this study, experiments were conducted to determine the stability of gene and protein expression in CTCs over a period of 96 h.ResultsBlood samples collected from healthy individuals and patients with cancer were each divided into five aliquots, which were stored at 2–8 °C and analyzed after 0, 24, 48, 72 and 96 h of storage. CTCs from patients and CD45-negative cells from healthy individuals were isolated each day using enrichment protocols, and qPCR was performed to determine expression levels of genes encoding specific biological markers. In addition, cells from breast and colon cancer cell lines were spiked into blood samples from healthy individuals, and these samples were stored and analyzed over a period of 96 h by PCR and by flow cytometry. The markers that were studied included housekeeping genes and genes associated with the response to chemotherapy, as well as genes encoding transcription factors. The results demonstrated that the expression profiles of specific genes and proteins in CTCs were not significantly affected by 72 h of storage. After 96 h of storage, expression of some genes was altered.ConclusionThe transportation of blood at low temperature (2–8 °C) in the presence of the anticoagulant EDTA can protect CTCs from alteration of gene and protein expression for at least 72 h. Furthermore, under these conditions, CTCs can be detected and isolated 96 h after blood collection.
Highlights
Minimizing the effects of transportation on the properties of biological material is a major challenge for the scientific community
Expression levels were measured in CD45-negative cells isolated from blood samples from two healthy individuals (Figs. 1, 2)
In the samples from one individual, gene expression did not alter significantly from 0 to 96 h, but in the samples from the second individual, quantitative polymerase chain reaction (qPCR) Ct values were significantly different after 96 h, but not after 72 h (p = 0.72), compared with the values at 0 h
Summary
Minimizing the effects of transportation on the properties of biological material is a major challenge for the scientific community. Detection and isolation of CTCs from blood samples is a routine activity in many laboratories, but concerns exist with regard to the maintenance of the cells during transportation. Experiments were conducted to determine the stability of gene and protein expression in CTCs over a period of 96 h. The detection and isolation of circulating tumor cells (CTCs) are useful procedures for cancer prognosis and prediction of treatment response. These cells constitute a subpopulation of cancer cells that have detached from the primary tumor and circulated. The ability to isolate CTCs from the samples is crucial, it is important to demonstrate RNA stability and maintenance of specific antigens. In addition to the detection of these cells, expression of relevant RNA and protein markers was evaluated
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