Detection of circulating tumour cells in blood by quantitative real-time RT-PCR: effect of pre-analytical time
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Ina H. Benoy
Abstract
Background: We and others have recently explored the use of quantitative real-time RT-PCR analysis for the detection of circulating tumour cells in blood of patients with breast cancer (BC). One major problem in these experiments is the in vitro instability of the cellular RNA. The copy number of mRNA can change during storage and transport at room temperature and this may hamper accurate quantitative measurements of specific transcripts, especially when working with small numbers of target mRNAs.
Methods: Peripheral blood samples were obtained from two healthy volunteers and 13 patients with BC. Blood was stored at room temperature for 0, 1, 2, 4, 6, 24, 48 and 72h. The potential alteration of gene expression for six target genes was investigated by quantitative real-time RT-PCR.
Results: For β-actin, GAPDH, cytokeratin-19 (CK-19) and HER2, a significant decrease in expression level occurs after 4h (CK-19 and HER2), 6h (β-actin) or 24h (GAPDH). Mammaglobin expression was only measurable in two samples and seems to be stable for at least 6h. For vascular endothelial growth factor (VEGF), a statistically significant increase in expression level is observed in samples processed 24h after collection.
Conclusions: Most transcripts were reduced in samples that were stored overnight at room temperature compared with fresh samples, but upregulation of transcripts, probably as an active response to cellular stress, also occurs when blood is removed from its in vivo environment and stored ex vivo. Optimally, blood samples and RNA should be processed or stabilised within 3h after collection to avoid interference of the in vivo gene expression signature by ex vivo stress responses.
Clin Chem Lab Med 2006;44:1082–7.
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©2006 by Walter de Gruyter Berlin New York
Articles in the same Issue
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Articles in the same Issue
- Point-of-care testing – can we move from anecdote to evidence?
- A long and winding road: defining the biological role and clinical importance of paraoxonases
- Point-of-care testing in the cardiovascular operating theatre
- Low-density lipoprotein receptor-related protein 5 and vitamin D receptor gene polymorphisms in relation to vitamin D levels in menopause
- The methylenetetrahydrofolate reductase C677T gene mutation is associated with hyperhomocysteinemia, cardiovascular disease and plasma B-type natriuretic peptide levels in Korea
- Exploring allelic imbalance within paraffin-embedded tumor biopsies using pyrosequencing technology
- Detection of circulating tumour cells in blood by quantitative real-time RT-PCR: effect of pre-analytical time
- Hereditary hyper-ACE-emia due to the Pro1199Leu mutation of somatic ACE as a potential pitfall in diagnosis: a first family outside Europe
- Molecular assay for detection of the common carnitine palmitoyltransferase 1A 1436(C>T) mutation
- Serum cytokine levels and the expression of estrogen and progesterone receptors in breast cancer patients
- Protein Z levels and prognosis in patients with acute coronary syndromes
- Determination of total bilirubin in whole blood from neonates: results from a French multicenter study
- Analysis of protein S-100B in serum: a methodological study
- Lipid peroxidation and homocysteine levels in Behçet's disease
- Lower expression of the α2,3-sialylated fibronectin glycoform and appearance of the asialo-fibronectin glycoform are associated with high concentrations of fibronectin in human seminal plasma with abnormal semen parameters
- Automated processing of whole blood samples for the determination of immunosuppressants by liquid chromatography tandem-mass spectrometry
- Role of methionine residues of albumin in T-R conversion of hemoglobin
- Rheumatoid factor interference in the determination of carbohydrate antigen 19-9 (CA 19-9)
- Significance of Elecsys® S100 immunoassay for real-time assessment of traumatic brain damage in multiple trauma patients
- IFCC primary reference procedures for the measurement of catalytic activity concentrations of enzymes at 37°C: International Federation of Clinical Chemistry and Laboratory Medicine (IFCC): Scientific Division, Committee on Reference Systems for Enzymes (C-RSE): Part 8. Reference procedure for the measurement of catalytic concentration of α-amylase: [α-Amylase: 1,4-α-D-glucan 4-glucanohydrolase (AMY), EC 3.2.1.1]
- ESEAP: the national External Quality Assessment Scheme for clinical chemistry in Greece and Cyprus
- Evaluation of whole-genome amplification using multiple-displacement amplification of a limited number of cells
- Comparison of various methods for the determination of total protein in urine
- Description of examinations and their results and ISO standard 15189
- Congress of Clinical Chemistry and Laboratory Medicine, Annual Congress of the Society of Clinical Chemistry and Laboratory Medicine (DGKL) in association with The Austrian Society for Laboratory Medicine and Clinical Chemistry, The Swiss Society for Clinical Chemistry (SGKC), The German Association of Technical Assistants in Medicine (dvta), Mannheim, Germany, October 1st - 4th, 2006
- First Congress of the Austrian Society for Laboratory Medicine and Clinical Chemistry (ÖGLMKC), Salzburg, October 4 – 7, 2006
