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PubMed1998

[Construction of the internal standard RNA and DNA templates for MRP gene quantitative RT-PCR analysis].

OBJECTIVE: To construct the internal standard RNA and DNA templates for quantitative reverse transcription-polymerase chain reaction (RT-PCR) and quantitatively measure the mRNA expression of MRP gene. METHODS AND RESULTS: A 292 bp fragment was amplified by PCR from plasmid pRC/RSV-MRP containing full-length MRP cDNA and inserted into the Sma I site of PUC19 vector, constructing a recombinant plasmid-pUMRP292. A 238 bp HGV fragment was amplified by PCR from plasmid pUHGV and inserted into the Cla I site of pUMRP292, constructing a new recombinant plasmid-pUMRP 292/HGV as the internal DNA competitive template for quantitative PCR of MRP. A 530 fragment containing the 292 bp of MRP and the 238 bp of HGV was cut down by EcoR I and Xba I pUMRP292/HGV and cloned into the transcriptional vector pSP72 by the same enzyme sites, constructing a recombinant plasmid-pSMRP292/HGV, and then pSMRP292/HGV was cleaved with EcoR V and transcripted in vitro by SP6 RNA polymerase, obtaining a 530 bp mutant MRP-RNA positive strand as the internal RNA competitive template for quantitative RT-PCR. CONCLUSION: The established quantitative RT-PCR assay is simple, rapid and sensitive, and the DNA competitive template is more simple, economic and reliable than the RNA one.

ATP-Binding Cassette Transporters↗

PubMed2005

Detecting cell-free circulating hTERT mRNA in the plasma may identify a subset of nonsmall cell lung cancer patients.

Human telomerase reverse transcriptase (hTERT), the catalytic subunity of telomerase, a marker of cell immortalization, is upregulated in most tumors, including nonsmall cell lung cancer (NSCLC). However, little is known about the role of assessing cell-free plasma circulating hTERT mRNA for tracing these tumors. We investigated by RT-polymerase chain reaction (PCR) and real-time quantitative PCR the prevalence and functional implications of hTERT mRNA in both tumor tissue and paired plasma samples in 34 (27 males and 7 females) stages I-IIIB NSCLC patients (21 adenocarcinomas and 13 squamous-cell carcinomas) by using intron- and exon-spanning primers. Plasma samples of ten healthy volunteers and normal lung tissue were used as negative controls. We detected hTERT mRNA in the plasma of 4 out of 34 (12%) tumor patients, but none was detected in the ten plasma samples of healthy volunteers. Normal lung tissue was completely devoid of hTERT mRNA. No association was found between hTERT plasma mRNA and clinicopathologic variables of the patients' population. We conclude that cell-free circulating hTERT mRNA is detectable in a subset of patients, whereas it is consistently absent in healthy volunteers. It can be added to the panel of multiple genetic tracers to detect lung cancer in the plasma of patients, although, per se, it is not specific for this tumor.

Adenocarcinoma↗

PubMed2005

Presence of Epstein-Barr virus in esophageal cancer is restricted to tumor infiltrating lymphocytes.

As representatives of low and high incidence countries respectively, 72 esophageal squamous cell carcinomas and 40 adenocarcinomas from Germany, and 43 esophageal squamous cell carcinomas from Russia were tested for the presence of Epstein-Barr virus (EBV) DNA by PCR and in situ hybridization. Thirty-four percent of the squamous cell carcinomas (SCC) and 26% of the adenocarcinomas (AC) contained EBV DNA as detected by nested PCR. Quantitative analysis using real time PCR revealed one copy of the EBV genome per every 27-200,000 cells. EBER RNA in situ hybridization showed no EBV-specific transcripts in the nuclei of the tumor cells. However, EBER transcripts were expressed in the nuclei of tumor infiltrating lymphocytes in 7 SCC and 1 AC of 24 EBV DNA positive cases. The present data provide no evidence for the persistence of EBV in the tumor cells of esophageal cancer. In contrast to a previous report from Taiwan, EBV is unlikely to play a role in esophageal carcinogenesis.

Adenocarcinoma↗

PubMed1991

Calpactin I in the differentiating embryonic chicken lens: mRNA levels and protein distribution.

Calpactin I, one of the EDTA-extractable proteins of the lens membrane, binds phospholipid and actin in a calcium-dependent manner. It is also known substrate of the pp60arc kinase. Analysis of embryonic chicken lens RNA with a bovine calpactin I-specific cDNA probe revealed the presence of a approximately 1.8 Kb calpactin mRNA in the lens cells. Six-day embryonic chicken lenses were microdissected into central epithelium, equatorial epithelium, and fiber cells. Total cytoplasmic RNA was isolated from these samples and calpactin I mRNA levels were determined by the polymerase chain reaction (PCR) following reverse transcription (RT). Quantitative PCR indicates that the calpactin I mRNA levels in the equatorial epithelium are greater than in the central epithelium by a factor of 12.7 +/- 2.7. Calpactin I mRNA in fiber cells is an additional 3.5 +/- 1.5 times greater than in the equatorial epithelium. Whole mounts of embryonic chicken lens epithelia and histological sections of whole lenses were also examined with an antibody directed against chicken calpactin I. Calpactin I was predominantly localized in a punctate distribution in equatorial epithelial cells and near the plasma membrane of elongate fiber cells. The elevated levels of calpactin I mRNA observed in the equatorial epithelium and fiber cells and the immunological localization of the protein suggest a possible role of calpactin I in the elongation of fiber cells during lens differentiation.

Animals↗

PubMed2006

Interleukin-1beta enhances the angiotensin-induced expression of plasminogen activator inhibitor-1 through angiotensin receptor upregulation in human astrocytes.

Plasminogen activator inhibitor-1 (PAI-1) regulates not only fibrinolysis but extracellular matrix remodeling, and angiotensin II is known to play an important role in controlling the expression of PAI-1 in astrocytes. We have studied the effect of interleukin-1beta (IL-1beta), one of major cytokines also active in the nervous system, on the angiotensin II-induced expression of PAI-1 in human astrocytes. Cultures of normal human astrocytes were stimulated with IL-1beta and angiotensin II, and the expression of mRNAs for angiotensin II type 1 receptor (AT1) and PAI-1 was analyzed by reverse transcription-polymerase chain reaction (RT-PCR) or real-time quantitative PCR. PAI-1 protein in astrocyte-conditioned medium was measured by enzyme-linked immunosorbent assay (ELISA). IL-1beta enhanced the expression of AT1 in astrocytes in time- and concentration-dependent manners. After 24-h stimulation, 10 ng/ml IL-1beta and 10 nM angiotensin II increased the levels of PAI-1 protein in astrocyte-conditioned medium by 1.9-fold and 1.8-fold of the basal value, respectively. There was no synergistic effect when the cells were stimulated simultaneously with IL-1beta and angiotensin II. When the cells were stimulated, with angiotensin II, 16 h after the stimulation with IL-1beta, the production of PAI-1 was enhanced by 1.4-fold as compared to the cells stimulated only with IL-1beta. CV-11794, an AT1 antagonist, inhibited the enhanced PAI-1 production in response to angiotensin II. We conclude that IL-1beta increases angiotensin II-induced PAI-1 secretion by astrocytes through the induction of AT1, and the enhanced secretion of PAI-1 may modulate functions of plasminogen activators in the nervous system.

Angiotensin II↗

PubMed2006

Expression of proteinase-activated receptors (PAR)-2 in articular chondrocytes is modulated by IL-1beta, TNF-alpha and TGF-beta.

OBJECTIVE: To investigate the modulation of expression of proteinase-activated receptor-2 (PAR-2) in articular chondrocytes by inflammatory cytokines. DESIGN: Articular synovium and cartilage tissues were collected from eight patients with osteoarthritis (OA), and three patients without arthropathy ("normal"). Chondrocytes were stimulated with interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha or transforming growth factor (TGF)-beta1. The expression of PAR-2 was detected using reverse transcriptase-polymerase chain reaction (PCR), Western blotting and immunofluorescence. Quantitative PCR was performed to assess the expression levels of PAR-2 messenger RNA (mRNA). RESULTS: The expression of PAR-2 mRNA was demonstrated in both OA and normal chondrocytes as well as in synovial fibroblasts. However, the level of PAR-2 in OA chondrocytes was much higher than in normal chondrocytes. Long-term culture revealed that PAR-2 mRNA expression was maintained up to three passages in OA but not in normal chondrocytes. IL-1beta and TNF-alpha both upregulated PAR-2 expression in normal and OA chondrocytes. In contrast, TGF-beta1 significantly decreased expression of PAR-2 in OA chondrocytes but increased PAR-2 in normal chondrocytes. CONCLUSIONS: Overexpression of PAR-2 in OA chondrocytes is upregulated by proinflammatory cytokines IL-1beta and TNF-alpha, and down-regulated by regulatory cytokine TGF-beta1. PAR-2 may be involved in the pathogenesis of OA.

Aged↗

PubMed2006

Human endothelial cells express NOD2/CARD15 and increase IL-6 secretion in response to muramyl dipeptide.

Mutations in the human NOD2/CARD15 gene cause Blau syndrome, an autoinflammatory disorder involving the joints, skin and eyes. Insights into the mechanism of this association may be gained by a further understanding of where NOD2 is expressed. The objective of this study was to analyze ocular endothelial cells for NOD2 expression. Human ocular tissue was analyzed by immunohistology using anti-NOD2 antisera. RNA isolated from iris, choroid and endothelial cell lines was analyzed by reverse transcription-PCR and real-time quantitative PCR. Gene regulation was studied by treating endothelial cells with TNF-alpha and IFN-gamma. Functional responses were assessed by measuring IL-6 release from endothelial cells treated with muramyl dipeptide (MDP), synthetic lipopeptide (Pam3CSK4) and lipopolysaccharide (LPS). Immunohistological analysis revealed staining of endothelial cells in the uveal tract. NOD2 expression was detected in primary ocular endothelial cell cultures, and levels increased in response to inflammatory cytokines. Endothelial cells from choroid demonstrated enhanced release of IL-6 in response to MDP, and synergy was observed following treatment with MDP and either Pam3CSK4 or LPS. The observations that endothelial cells express NOD2, upregulate NOD2 in response to stimuli known to promote NOD2 expression and show synergistic cytokine responses to MDP and TLR ligands previously shown to be mediated by NOD2 are informative since they may be relevant to pathogenic mechanisms leading to the spectrum of inflammation seen in Blau syndrome.

Acetylmuramyl-Alanyl-Isoglutamine↗

PubMed2005

Spinal phospholipase A2 in inflammatory hyperalgesia: role of group IVA cPLA2.

Current work has shown the importance of spinal cyclooxygenase (COX) products in facilitatory processes leading to tissue injury induced hyperalgesia. This cascade must originate with free arachidonic acid (AA) released by the activity of spinal phospholipase A2s (PLA2). In the present work, we studied the role of PLA2s in spinal sensitization. We first demonstrate the presence of constitutive mRNA in the spinal cord for PLA2 Groups IB, IIA, IIC, IVA, V and VI by reverse transcription-polymerase chain reaction (RT-PCR) and sequencing. Using quantitative-PCR, we found that Group IVA cPLA2 and Group VI iPLA2 are the predominant PLA2 messages in the spinal cord. Western blotting and activity assays specific for Group IVA cPLA2 and Group VI iPLA2 verified the presence of these enzymes. PLA2 activity in spinal cord homogenates was suppressed by methyl arachidonyl fluorophosphonate (MAFP) and arachidonyl trifluoromethylketone (AACOCF3), mixed inhibitors of Group IVA cPLA2 and Group VI iPLA2 as well as by bromoenol lactone (BEL), a Group VI iPLA2 inhibitor. The spinal expression of PLA2 mRNA or protein was not altered in the face of peripheral inflammation. Secondly, we showed that intrathecal (i.t.) administration of MAFP and AACOCF3, but not BEL, dose-dependently prevented thermal hyperalgesia induced by intraplantar carrageenan as well as formalin-induced flinching. Finally, i.t. injection of AACOCF3, at antihyperalgesic doses, decreased the release of prostaglandin E2 (PGE2) into spinal dialysate evoked by i.t. NMDA, while i.t. injection of BEL had no effect. Taken together, this work points to a role for constitutive Group IVA cPLA2 in spinal nociceptive processing.

Animals↗

PubMed2006

The arginine regulon of Escherichia coli: whole-system transcriptome analysis discovers new genes and provides an integrated view of arginine regulation.

Analysis of the response to arginine of the Escherichia coli K-12 transcriptome by microarray hybridization and real-time quantitative PCR provides the first coherent quantitative picture of the ArgR-mediated repression of arginine biosynthesis and uptake genes. Transcriptional repression was shown to be the major control mechanism of the biosynthetic genes, leaving only limited room for additional transcriptional or post-transcriptional regulation. The art genes, encoding the specific arginine uptake system, are subject to ArgR-mediated repression, with strong repression of artJ, encoding the periplasmic binding protein of the system. The hisJQMP genes of the histidine transporter (part of the lysine-arginine-ornithine uptake system) were discovered to be a part of the arginine regulon. Analysis of their control region with reporter gene fusions and electrophoretic mobility shift in the presence of pure ArgR repressor showed the involvement in repression of the ArgR protein and an ARG box 120 bp upstream of hisJ. No repression of the genes of the third uptake system, arginine-ornithine, was observed. Finally, comparison of the time course of arginine repression of gene transcription with the evolution of the specific activities of the cognate enzymes showed that while full genetic repression was achieved 2 min after arginine addition, enzyme concentrations were diluted at the rate of cell division. This emphasizes the importance of feedback inhibition of the first enzymic step in the pathway in controlling the metabolic flow through biosynthesis in the period following the onset of repression.

Amino Acid Transport Systems, Basic↗

PubMed2006

Widespread occurrence of phage-encoded exotoxin genes in terrestrial and aquatic environments in Southern California.

Many human diseases are caused by pathogens that produce exotoxins. The genes that encode these exotoxins are frequently encoded by mobile DNA elements such as plasmids or phage. Mobile DNA elements can move exotoxin genes among microbial hosts, converting avirulent bacteria into pathogens. Phage and bacteria from water, soil, and sediment environments represent a potential reservoir of phage- and plasmid-encoded exotoxin genes. The genes encoding exotoxins that are the causes of cholera, diphtheria, enterohemorrhagic diarrhea, and Staphylococcus aureus food poisoning were found in soil, sediment, and water samples by standard PCR assays from locations where the human diseases are uncommon or nonexistent. On average, at least one of the target exotoxin genes was detected in approximately 15% of the more than 300 environmental samples tested. The results of standard PCR assays were confirmed by quantitative PCR (QPCR) and Southern dot blot analyses. Agreement between the results of the standard PCR and QPCR ranged from 63% to 84%; and the agreement between standard PCR and Southern dot blots ranged from 50% to 66%. Both the cholera and shiga exotoxin genes were also found in the free phage DNA fraction. The results indicate that phage-encoded exotoxin genes are widespread and mobile in terrestrial and aquatic environments.

Bacterial Infections↗

PubMed2006

Glycoprotein 340 in normal human ocular surface tissues and tear film.

The tear film is a complex mixture of secreted fluid, ions, proteins, glycoproteins, and lipids that lubricates and protects the ocular surface. Recently, several antimicrobial peptides have been described in the tear fluid. In this study, we describe the presence of the large secreted glycoprotein gp340 in the tear film. Western blot analysis showed that gp340 is abundant in secreted tears and in the lacrimal glands. Lesser amounts of gp340 were detected in the cornea and conjunctiva. Consistent with Western blot data, reverse transcription-PCR and real-time quantitative PCR showed that gp340 transcripts were abundant in lacrimal gland tissue and were also present in the cornea and conjunctiva. Immunohistochemistry localized gp340 to the acinar cells of the lacrimal gland and the deeper layers of the conjunctival epithelium. gp340 was not detected in conjunctival goblet cells. In the cornea, gp340 was present only in a peripheral band of basal epithelial cells, suggesting that gp340 may play a role in the cycle of corneal epithelial renewal. To determine if tear film gp340 may function as a bacterial agglutinin as it does in saliva, tears were incubated with streptococcal cells and the formation of bacterial aggregates was monitored. Addition of tears to late-exponential-phase Streptococcus mutans cells resulted in time- and dose-dependent aggregation of the bacteria. Furthermore, Western blot analysis confirmed the presence of cell-associated gp340 in isolated bacterial aggregates. The ocular pathogen Staphylococcus aureus, but not Pseudomonas aeruginosa, also aggregated when incubated with tears. These results suggest that gp340 is a normal component of the tear film and that the glycoprotein may function as a bacterial agglutinin.

Adult↗

PubMed1999

Quantification of human cytomegalovirus DNA in amniotic fluid of mothers of congenitally infected fetuses.

A quantitative PCR assay was used to quantitate human cytomegalovirus DNA in amniotic fluid of mothers of 21 fetuses with congenital infection. Seven fetuses presented ultrasound abnormalities or were born with symptoms, whereas 14 fetuses were subclinically infected. Although the median DNA level was higher in symptomatic fetuses, the difference was not statistically significant (P = 0.09).

Amniotic Fluid↗

PubMedDate not supplied

Adenoviral mediated anti-sense CD38 attenuates TNF-alpha-induced changes in calcium homeostasis of human airway smooth muscle cells.

CD38 is a membrane-bound protein involved in the synthesis and degradation of cyclic-ADP-ribose (cADPR). cADPR mobilizes calcium from intracellular stores in airway smooth muscle cells. To determine the role of CD38/cADPR signaling in calcium regulation in human airway smooth muscle (HASM) cells, we downregulated CD38 expression using a recombinant replication-defective adenovirus with anti-sense human CD38 (Ad-asCD38). CD38 expression was determined by RT-PCR and real-time quantitative PCR, and ADP-ribosyl cyclase (cyclase) activity was determined by competitive binding assay. In HASM cells infected with Ad-asCD38, TNF-alpha-induced, augmented-CD38 expression and cyclase activity were significantly lower than in TNF-alpha-treated cells. The net intracellular calcium responses to 10 nmol/L bradykinin were measured in HASM cells by fluorescence imaging. In cells infected with Ad-asCD38 in the presence of TNF-alpha, the net intracellular Ca2+ responses were significantly lower than in cells treated with TNF-alpha in the presence of the control vector (p < 0.001). These results provide evidence for the feasibility of using adenoviral vectors for gene transfer to down regulate gene expression, and confirm the role of CD38 in calcium homeostatis in ASM cells.

ADP-ribosyl Cyclase↗

PubMed2005

Expression of ADAM15 in rheumatoid synovium: up-regulation by vascular endothelial growth factor and possible implications for angiogenesis.

ADAMs (a disintegrin and metalloproteinases) comprise a new gene family of metalloproteinases, and may play roles in cell-cell interaction, cell migration, signal transduction, shedding of membrane-anchored proteins and degradation of extracellular matrix. We screened the mRNA expression of 10 different ADAMs with a putative metalloproteinase motif in synovial tissues from patients with rheumatoid arthritis (RA) or osteoarthritis (OA). Reverse transcription PCR and real-time quantitative PCR analyses indicated that among the ADAMs, ADAM15 mRNA was more frequently expressed in the RA samples and its expression level was significantly 3.8-fold higher in RA than in OA (p < 0.01). In situ hybridization, immunohistochemistry and immunoblotting demonstrated that ADAM15 is expressed in active and precursor forms in the synovial lining cells, endothelial cells of blood vessels and macrophage-like cells in the sublining layer of RA synovium. There was a direct correlation between ADAM15 mRNA expression levels and vascular density in the synovial tissues (r = 0.907, p < 0.001; n = 20). ADAM15 was constitutively expressed in RA synovial fibroblasts and human umbilical vein endothelial cells (HUVECs), and the expression level was increased in HUVECs by treatment with vascular endothelial growth factor (VEGF)165. On the other hand, ADAM15 expression in RA synovial fibroblasts was enhanced with VEGF165 only if vascular endothelial growth factor receptor (VEGFR)-2 expression was induced by treatment with tumor necrosis factor-alpha, and the expression was blocked with SU1498, a specific inhibitor of VEGFR-2. These data demonstrate that ADAM15 is overexpressed in RA synovium and its expression is up-regulated by the action of VEGF165 through VEGFR-2, and suggest the possibility that ADAM15 is involved in angiogenesis in RA synovium.

ADAM Proteins↗

PubMed2004

Cloning of the cDNA for thyroid stimulating hormone beta subunit and changes in activity of the pituitary-thyroid axis during silvering of the Japanese eel, Anguilla japonica.

The purposes of this study were: (1) to clone the cDNA encoding pituitary thyroid-stimulating hormone beta subunit (TSH beta) of the Japanese eel, Anguilla japonica, together with its genomic DNA sequence, for phylogenetic analysis, and to study the regulation of the TSH beta gene expression in cultured pituitaries; and (2) to investigate the transcript levels of pituitary TSH beta mRNA and the serum thyroxine profiles at different stages of ovarian development before and during silvering in the wild female eels. The maturity of female eels was divided into four stages, juvenile, sub-adult, pre-silver, and silver, based on skin color and oocyte diameter. The genomic DNA of the TSH beta subunit contains two introns and three exons, and the TSH beta protein possesses a putative signal peptide of 20 amino acids and a mature peptide of 127 amino acids. The amino acid sequence identities of TSH beta mature peptide of Japanese eel compared with those of teleosts and other vertebrates are: European eel (98.4%), salmonids (60.6-61.3%), carps (52.0-56.7%), sturgeon (48.4%), and tetrapods (42.9-45.2%). In in vitro studies of the regulation of TSH beta mRNA it was found that thyrotropin-releasing hormone increased while thyroxine decreased its expression. RT-PCR and real-time quantitative PCR analysis showed that the transcript levels of TSH beta subunit increased during eel silvering. The serum thyroxine levels also increased in parallel with TSH beta mRNA expression during silvering, supporting the hypothesis that the hypothalamus-pituitary-thyroid axis is correlated to silvering in the wild female Japanese eels.

Amino Acid Sequence↗

PubMed1997

Inhibition of PCR amplification by a point mutation downstream of a primer.

A T-->C point mutation is shown to specifically inhibit PCR amplification when compared to wild-type controls in exon H of the factor IX gene. Multiple primers of different lengths and locations were designed to examine this phenomenon. The experiments suggest that poor annealing and/or extension from the downstream primer are responsible for the observed inhibition and that the mutation can exert an inhibitory effect upon PCR amplification at a distance of at least 84 bp. The inhibition was not alleviated when amplification conditions such as annealing temperature, time of extension, type of DNA polymerase or concentration of DNA template, primer or DNA polymerase were varied. The inhibitory factor(s) are likely to be contained within the amplified segment itself because neither the use of a previously amplified PCR product as template for nested PCRs nor the restriction enzyme digestion of that previously amplified product relieved the inhibition of PCR amplification in the mutant sample. Computer analyses with the FOLDRNA and FOLDDNA programs did not reveal the mechanism of inhibition. Although dramatic inhibition, as shown here, may be uncommon, more subtle inhibition may be frequent. Documentation of differential amplification caused by a single-base substitution in template sequence has implications for certain commonly used PCR-based methods such as quantitative PCR, differential display and DNA fingerprinting. In addition, heterozygous single-base pair mutations down-stream of a primer may be missed if the PCR is inhibited; alternatively; the mutation may appear to be homozygous if amplification of the mutated allele is selectively enhanced.

DNA↗

PubMed2001

Effects of aflastatin A, an inhibitor of aflatoxin production, on aflatoxin biosynthetic pathway and glucose metabolism in Aspergillus parasiticus.

Aflastatin A inhibits aflatoxin production by Aspergillus parasiticus via an unknown mechanism. We found that aflastatin A clearly inhibited production of norsolorinic acid, an early biosynthetic intermediate of aflatoxin, at a concentration of 0.25 microg/ml. Reverse-transcriptase polymerase chain reaction (RT-PCR), and real-time quantitative PCR (TaqMan PCR) experiments indicated that the transcription of genes encoding aflatoxin biosynthetic enzymes (pksA, ver-1, and omtA) and a gene encoding a regulatory protein for expression of the biosynthetic enzymes (aflR) were significantly reduced by the addition of aflastatin A. We also found that aflastatin A elevated the glucose consumption and ethanol accumulation by A. parasiticus, and repressed transcription of genes involved in ethanol utilization. These results suggest that aflastatin A inhibits a very early step in aflatoxin biosynthesis prior to the transcription of aflR and can influence glucose metabolism in the fungus.

Aflatoxins↗

PubMedDate not supplied

Difference between free circulating plasma and serum DNA in patients with colorectal liver metastases.

BACKGROUND: Previous studies suggest that the amount of circulating cell-free DNA in plasma or serum may be a promising marker for diagnosis and prediction of prognosis in patients with tumours. However, it is unclear whether DNA from plasma or from serum better reflects the clinical status. PATIENTS AND METHODS: To investigate the possible difference between serum DNA and plasma DNA levels, blood was collected from 26 patients with liver metastases and 28 age-matched healthy donors. The patients had clinical follow-up after surgery (median 23 months). DNA was quantified by two independent methods: real-time quantitative PCR (TaqMan) and fluorimetric DNA quantitation (Picogreen reagent). RESULTS: Serum DNA and plasma DNA levels did not correlate and each had a different relationship with diagnosis and prognosis. Only serum DNA was significantly associated with the presence of liver metastases, whereas only plasma DNA was predictive for recurrences. Typically, serum DNA amounts were higher than plasma in 24 out of 26 patients. CONCLUSION: This difference between plasma and serum reflects an in vitro process that occurs during clotting. High DNA increase in serum represents an indirect, yet tumour-related process. Plasma DNA levels better represent in vivo levels of circulating DNA. This has implications forfuture studies.

Aged↗