circulating nucleic acids -- RNA - DNA - microRNA - small RNAs  (1)
circulating nucleic acids - focus on microRNA  (2)
circulating nucleic acids - new papers  (3)
Liquid Biopsy


Most of the DNA and RNA in the body is located within cells, but a small amount of nucleic acids can also be found circulating freely in the blood. These DNA, RNA and small RNA molecules are thought to come from dying cells that release their contents into the blood as they break down. The term "Circulating Nucleic Acids =CNA" refers to segments of DNA or RNA found in the bloodstream.

CNAs offers a non-invasive approach to a wide range in diagnostics of clinical disorders that will allow the basic information necessary not only for use in predictive medicine but also for direct use in acute medicine. Further free CNAs offer unique opportunities for early diagnosis of clinical conditions, e.g. in early cancer detection.

Although DNA was first demonstrated in human blood from healthy donors, pregnant women and clinical patients in 1948, the structure of DNA was still to be determined as was the elucidation of its role as the basis of the gene. Consequently, no interest was shown in the presence of DNA in the circulatory system until high DNA levels were demonstrated in the blood of patients with systemic lupus erythematosus. Similar observations were also made in acute medicine, diabetes, oncology and fetal medicine. The presence of DNA and RNA in plasma of patients has been recognised since the 1970s - see table.

Where does the free CNA come from - The nucleic acid sources:
  • DNA
There are several possible sources of blood DNA, namely:
  • break down of blood cells
  • brake down of any pathogens, e.g. bacteria or viruses
  • leucocyte surface DNA
  • apoptosis
  • necrosis
  • spontaneous release of a newly synthesised DNA
  • spontaneous release of DNA/RNA-lipoprotein complex from healthy cells
Only small amounts of DNA are yielded by the first two possibilities with just nasopharyngeal carcinoma Barr virus and human papilloma virus carcinoma DNAs having been identified and the breakdown of bacteria and bloods cells yielding only low levels of DNA.
The nucleic acid in question can be RNA, mitochondrial DNA or genomic DNA, but DNA is generally used as it is less labile than RNA.
Necrosis is clearly an option for the origin of Circulating Nucleic Acids in Plasma and Serum (CNAPS). However, when the double stranded CNAPS DNA is separated by gel electrophoresis, the fragments tend to form a ladder rather than a smear. The ladder fragments are mainly 180 - 1,000 bp in size and so are likely to be formed by apoptosis. DNA released by necrosis is incompletely and non-specifically digested and so smears on electrophoretic separation due to its fragment sizes of about 10,000bp; this is not a major source of CNAPS.
Apoptosis is confirmed as a major DNA source especially since nucleosomes are present in the blood e.g. of cancer patients. Naked DNA fragments are also found in serum, possibly due to apoptosis.
  • RNA
RNA is only recently of importance through its exploitation in clinical diagnosis and prognosis. The stability of RNA or microRNA in the bloodstream is due to the availability and type of the RNAs and RNAses present.
However, a newly synthesised RNA is released spontaneously from cells together with the DNA-lipoprotein complex. In consequence, RNA is primarily released by apoptosis and through the DNA/RNA-lipoprotein complex. Some RNA may also be derived by necrosis e.g. some mRNAs.
  • microRNA
MicroRNAs (miRNAs) are evolutionarily conserved, endogenous, noncoding small RNAs that act as post-transcriptional gene regulators. Experimental evidence has shown that miRNAs can play roles as oncogenes or tumor suppressor genes, suggesting their contribution to cancer development and progression. Expression profiles of human miRNAs demonstrated that many miRNAs are deregulated in cancers and are differentially expressed in normal tissues and cancers. Therefore, miRNA profiling is used to create signatures for a variety of cancers, indicating that the profile will help further establish molecular diagnosis, prognosis and therapy using miRNAs. It was shown that  the aberrant expression of miRNAs in human cancer, and discusses the potential of these miRNAs as biomarkers and targets/molecules for molecular therapy.
  • CNA as biomarkers in various cancer types
A range of markers have been proposed for the identification of a particular cancer, though there is frequent conflict in the literature as to the effectiveness of particular probes. However, recently, hypermethylated CpG in the promotor region of tumour suppressor genes has been suggested to trigger local gene silencing. Aberrant methylation of the p26 tumour suppressor gene was the first to be detected in liver, breast and lung cancer.
Nucleic acids can be found in small amounts in healthy and diseased human plasma/serum. Higher concentrations of DNA are present in the plasma of cancer patients sharing some characteristics with DNA of tumor cells. Together with decreased strand stability, the presence of specific oncogene or tumor-suppressor gene mutations, microsatellite alterations, Ig rearrangements and hypermethylation of several genes may be detected. Moreover, tumor-related mRNA has been found circulating in the plasma/serum.
  • Conclusion
The results obtained in many different cancers have opened a new research area indicating that circulating nucleic acids might eventually be used for the development of noninvasive diagnostic, prognostic and follow-up tests for cancer.
  • References

Cell-free microRNAs: potential biomarkers in need of standardized reporting
Michaela B. Kirschner, Nico van Zandwijk and Glen Reid
Asbestos Diseases Research Institute, University of Sydney, Sydney, NSW, Australia
Front. Genet., 19 April 2013

MicroRNAs are abundantly present and surprisingly stable in multiple biological fluids. These findings have been followed by numerous reverse transcription real-time quantitative PCR (RT-qPCR)-based reports revealing the clinical potential of using microRNA levels in body fluids as a biomarker of disease. Despite a rapidly increasing body of literature, the field has failed to adopt a set of standardized criteria for reporting the methodology used in the quantification of cell-free microRNAs. Not only do many studies based on RT-qPCR fail to address the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) criteria but frequently there is also a distinct lack of detail in descriptions of sample source and RNA isolation. As a direct result, it is often impossible to compare the results of different studies, which in turn, hinders progress in the field. To address this point, we propose a simple set of criteria to be used in conjunction with MIQE to reveal the true potential of cell-free microRNAs as biomarkers.

Circulating Nucleic Acids in Plasma and Serum - An Overview
Annals of the New York Academy of Sciences 2001

The recent interest in nucleic acids in plasma and serum has opened up numerous new areas of investigation and new possibilities for molecular diagnosis. In oncology, tumor-derived genetic changes, epigenetic alterations, and viral nucleic acids have been found in the plasma/serum of cancer patients. These findings have important implications for the detection, monitoring, and prognostication of many types of malignancies. In prenatal diagnosis, the discovery of fetal DNA in maternal plasma and serum has provided a noninvasive source of fetal genetic material for analysis. This development has important implications for the realization of noninvasive prenatal diagnosis and has provided new methods for the monitoring of pregnancy-associated disorders. Plasma DNA technology has also found recent applications in the fields of organ transplantation, posttrauma monitoring, and infectious agent detection. Future areas of study include circulating RNA in plasma and the elucidation of the biology of release, clearance, and possible functionality of plasma nucleic acids.

Circulating DNA - a new diagnostic gold mine?
Ziegler A, Zangemeister-Wittke U, Stahel RA.
Cancer Treat Rev. 2002 Oct;28(5):255-71.

The recent discovery that cell-free DNA can be shed into the bloodstream as a result of tumour cell death has generated great interest. Numerous studies have demonstrated tumour-specific alterations in DNA recovered from plasma or serum of patients with various malignancies, a finding that has potential for molecular diagnosis and prognosis. The implication is that tumour-derived nucleic acids of human or viral origin can be retrieved from blood by a minimally invasive procedure, and used as a surrogate tumour marker to monitor the course of the disease or aid in early diagnosis. The present review will describe the main areas of ongoing investigation, with particular emphasis on technical issues and available data of clinical relevance.

Circulating (cell-free) nucleic acids - a promising, non-invasive tool for early detection of several human diseases.
Swarup V, Rajeswari MR.
FEBS Lett. 2007 581(5): 795-799

Circulating nucleic acids (CNA) are present in small amounts in the plasma of healthy individuals. However, increased levels of plasma CNA have been reported in a number of clinical disorders like cancer, stroke, trauma, myocardial infarction, autoimmune disorders, and pregnancy-associated complications. CNA has received special attention because of its potential application as a non-invasive, rapid and sensitive tool for molecular diagnosis and monitoring of acute pathologies and the prenatal diagnosis of fetal genetic diseases. This review throws light on the current status of blood CNA as a diagnostic marker and its potential as a powerful tool in the future.

Diagnostic developments involving cell-free (circulating) nucleic acids.
Tong YK, Lo YM.
Clin Chim Acta. 2006 363(1-2): 187-196

BACKGROUND: The detection of circulating nucleic acids has long been explored for the non-invasive diagnosis of a variety of clinical conditions. In earlier studies, detection of circulating DNA has been investigated for the detection of various forms of cancer. Metastasis and recurrence in certain cancer types have been associated with the presence of high levels of tumor-derived DNA in the circulation. In the case of pregnancies, detection of fetal DNA in maternal plasma is a useful tool for detecting and monitoring certain fetal diseases and pregnancy-associated complications. Similarly, levels of circulating DNA have been reported to be elevated in acute medical emergencies, including trauma and stroke, and have been explored as indicators of clinical severity. Apart from circulating DNA, much attention and effort have been put into the study of circulating RNA over the last few years. This area started from the detection of tumor-derived RNA in the plasma of cancer patients. Soon after that, detection of circulating fetal RNA in maternal plasma was described. Plasma RNA detection appears to be a promising approach for the development of gender- and polymorphism-independent fetal markers for prenatal diagnosis and monitoring. This development also opens up the possibility of non-invasive prenatal gene expression profiling by maternal blood analysis. Besides circulating DNA and RNA in plasma and serum, cell-free DNA in other body fluids, such as urine, has been detected in patients with different clinical conditions. Regardless of the sources of cell-free DNA for clinical use, the amount is frequently scarce.
METHODS: Technical advancements in detecting free DNA have been made over the years.
CONCLUSIONS: It is likely that further developments in the field of circulating nucleic acids will provide us with new diagnostic and monitoring possibilities over the next few years.

Plasma nucleic acids in the diagnosis and management of malignant disease.
Johnson PJ, Lo YM.
Clin Chem. 2002 48(8): 1186-1193. Review.

BACKGROUND: There is a need for development of molecular markers of cancer that can be used clinically for the detection, prognostication, and monitoring of cancer. Recently, there has been much interest in the potential use of nucleic acid markers in plasma and serum for this purpose.
APPROACH: We reviewed published literature up to 2002 on the topic, with a particular emphasis on reports published between 1996 and 2002.
CONTENT: The nucleic acid markers described in plasma and serum include oncogene mutations/amplifications, microsatellite alterations, and gene rearrangements. Such markers have been described in many cancer types, including lung, colon, and breast. Epigenetic alterations, such as aberrant promoter methylation, have been identified in plasma and serum. Viral nucleic acid markers, such as Epstein-Barr virus DNA in plasma and serum, are reviewed in detail with regard to their application to virus-associated cancers such as nasopharyngeal carcinoma and various lymphomas. More recently, mitochondrial DNA and tumor-related mRNAs have been identified in plasma and serum from patients with several types of tumors.
CONCLUSIONS: Circulating nucleic acids are an emerging class of molecular tumor markers. Their wide applicability and clinical relationship with the malignant state will likely grant them increasing clinical importance in the near future.

Chemical modification resolves the asymmetry of siRNA strand degradation in human blood serum.
Hoerter JA, Walter NG
RNA. 2007 13(11): 1887-1893.

Small interfering (si)RNAs have recently been used to therapeutically silence genes in vivo after intravenous systemic delivery. Further progress in the development of siRNA therapeutics will in part rely on tailoring site-specific chemical modifications of siRNAs to optimize their pharmacokinetic properties. Advances are particularly needed to improve the nucleolytic stability of these double-stranded RNA drugs in vivo and suppress adverse off-target gene silencing effects. Here we demonstrate that specific chemical 2'-O-methylation, which has already been shown to ameliorate the omnipresent off-target toxicity of siRNAs, selectively protects the particularly vulnerable 5'-end of the guide strand against exonucleolytic degradation in human blood serum. Specific chemical modification thus resolves the asymmetric degradation of the guide and passenger strands, which is inherent to the thermodynamic asymmetry of the siRNA termini as required for proper utilization of the guide strand in RNA interference.

Circulating tumour-derived DNA and RNA markers in blood: a tool for early detection, diagnostics, and follow-up?
Bremnes RM, Sirera R, Camps C.
Lung Cancer 2005 49(1): 1-12. Review.

BACKGROUND: Lung cancer is the most common cause of cancer death in developed countries. The prognosis is poor with only 10-15% of patients surviving 5 years after diagnosis. This dismal prognosis is attributed to the lack of efficient diagnostic methods for early detection and lack of successful treatment for metastatic disease. Within the last decade, rapid advances in molecular biology and radiology have provided a rational basis for improving early detection and patients' outcome. A non-invasive blood test effective in detecting preneoplastic changes or early lung cancer in high risk individuals has been perceived as a holy grail by cancer researchers.
METHODS: The introduction of polymerase chain reaction (PCR)-based technology in the late 1980s and its refinement over the last 10 years have allowed us to detect and quantify extremely small amounts of tumour-derived nucleic acids. This has led to an increased knowledge of the molecular pathogenesis of lung cancer and a basis for the use of DNA and RNA markers in blood for early cancer detection, diagnostics, and follow-up. Common genetic alterations in lung carcinogenesis are already well known. We reviewed published literature on DNA and RNA in plasma or serum in lung cancer patients up to 2004, with particular emphasis on reports published since 1995.
RESULTS: Twenty-two clinical studies have evaluating the role of DNA and RNA aberrations in the blood of lung cancer patients. A total of 1618 (range 10-163/study) cases and 595 (range 10-120/study) control cases were evaluated, and overall plasma/serum abnormalities were found in 43% (range 0-78%) of cases and 0.8% of healthy controls. For (1) total DNA and gene expression levels, 61% (range 53-71%) of cases and 0.9% of controls; (2) oncogene mutations, 16% (range 0-30%) and 0%; (3) microsatellite alterations, 46% (range 24-71%) and 21% (controls with non-malignant pulmonary disease); (4) promoter methylation, 42% (range 5-73%) and 0%; (5) tumour-related RNAs, 54% (range 39-78%) and 6%. In general, the studies contain small series of lung cancer patients and even smaller or missing case control groups.
CONCLUSION: The analysis of circulating DNA or RNA in plasma is a promising non-invasive diagnostic tool, requiring only a limited blood sample. Its wide applicability and potential importance will possibly lead to increasing clinical impact in the near future. However, large prospective clinical studies are needed to validate and standardise any tests for DNA or RNA alteration in plasma or serum of high risk individuals or patients with established lung cancer.

Clinical Relevance of Circulating Nucleosomes in Cancer
Stefan Holdenrieder , Dorothea Nagel , Andreas Schalhorn, Volker Heinemann, Ralf Wilkowski, Joachim von Pawel, Hannelore Raith, Knut Feldmann, Andreas E. Kremer, Susanne Müller, Sandra Geiger, Gerhard F. Hamann, Dietrich Seidel, and Petra Stieber
Annals of the New York Academy of Sciences (2008) 1137
Issue Circulating Nucleic Acids in Plasma and Serum V, Pages 180 - 189

Nucleosomes, complexes of DNA and histone proteins, are released during cell death into the blood circulation. Elevated serum and plasma levels have been found in various forms of cancer, but also in autoimmune diseases and acute situations such as stroke, trauma, and during sepsis. Here, the clinical relevance of circulating nucleosomes for diagnosis, staging, prognosis, and therapeutic monitoring of cancer is reviewed. Several studies have shown that levels of nucleosomes are significantly higher in serum and plasma of cancer patients in comparison to healthy controls. However, because of elevations of nucleosome levels in patients with benign diseases relevant for differential diagnosis, they are not suitable for cancer diagnosis. Concerning tumor staging, nucleosome levels correlate with tumor stage and presence of metastases in gastrointestinal cancer, but not in other tumor types. Prognostic value of circulating nucleosomes is found in lung cancer in univariate analyses, but not in multivariate analyses. Circulating nucleosomes are most informative for the monitoring of cytotoxic therapy. Strongly decreasing levels are mainly found in patients with remission of disease, whereas constantly high or increasing values are associated with progressive disease during chemo- and radiotherapy. In addition, therapy outcome is already indicated by the nucleosomal course during the first week of chemo- and radiotherapy in patients with lung, pancreatic, and colorectal cancer as well as in hematologic malignancies. Despite their non-tumor-specificity, kinetics of nucleosomes are valuable markers for the early estimation of therapeutic efficacy and may be helpful to adapting early cancer therapy in the future.

Circulating nucleic acids in plasma or serum.
P. Anker, J. Lyautey, C. Lederrey and M. Stroun
Clinica Chimica Acta Volume 313, Issues 1-2, November 2001, 143-146

Nucleic acids can be found in small amounts in healthy and diseased human plasma/serum. Higher concentrations of DNA are present in the plasma of cancer patients sharing some characteristics with DNA of tumor cells. Together with decreased strand stability, the presence of specific oncogene or tumor-suppressor gene mutations, microsatellite alterations, Ig rearrangements and hypermethylation of several genes may be detected. Moreover, tumor-related mRNA has been found circulating in the plasma/serum. CONCLUSIONS: The results obtained in many different cancers have opened a new research area indicating that circulating nucleic acids might eventually be used for the development of noninvasive diagnostic, prognostic and follow-up tests for cancer.

Cell-free DNA in serum and plasma: comparison of ELISA and quantitative PCR.
Holdenrieder S, Stieber P, Chan LY, Geiger S, Kremer A, Nagel D, Lo YM.
Clin Chem. 2005 51(8): 1544-1546

Although circulating DNA has generally been referred to as cell-free DNA, it is likely that a significant proportion is bound to protein molecules, possibly as nucleosomes. This conclusion is supported by theory and by observations. Theoretically, circulating DNA is mostly released from degrading cells after cleavage by endonucleases that cut the chromatin into the basic nucleosomal elements (1)(2). Empirically, DNA fragments in circulation are mainly sized in multiples of the nucleosomal DNA (3)(4). Filtration experiments have shown that circulating RNA seems to be associated with particles, whereas DNA is not (5). This might be attributable to the arrangement of DNA in nucleosomes, which conserves them from proteolytic digestion in blood. Nucleosomal complexes consist of duplicate copies of the histones H2A, H2B, H3, and H4 as core proteins, with ~146 bp of DNA on the outside (6).
In various pathologic conditions, qualitative and quantitative changes in circulating DNA have been shown. Only small amounts of serum or plasma DNA have been observed in healthy individuals, whereas high concentrations have been described in patients with various malignancies and in those with several benign diseases, such as infections, sepsis, trauma, stroke, and autoimmune diseases (3)(7)(8)(9)(10)(11)(12)(13). Because most of these disorders are associated with increased rates of cell death events, from either apoptosis or necrosis, these mechanisms are considered to be the main sources for circulating DNA. Active release of DNA by lymphocytes is thought to be of minor relevance (2)(14)(15)(16). In cancer patients during chemo- and radiotherapy, the kinetics of circulating DNA correlated with tumor response to therapy and also with posttherapeutic tumor recurrence (13)(17)(18)(19). Recently, it was shown that initial changes in nucleosomal DNA during chemotherapy are predictive of therapeutic efficacy (19)....................

Fetal nucleic acids in maternal plasma.
YM Dennis Lo
Ann N Y Acad Sci (2008) 1137: 140-143

The discovery of cell-free fetal nucleic acids in maternal plasma has opened up new possibilities for noninvasive prenatal diagnosis. Over the last few years, a number of approaches have been demonstrated to allow such circulating fetal nucleic acids to be used for the prenatal detection of chromosomal aneuploidies. One such approach involves the enrichment of fetal DNA, such as by size fractionation or by the controversial formaldehyde treatment technique. A second approach involves the targeting of fetal-specific nucleic acid molecules, including fetal-specific epigenetic markers and placenta-specific mRNA markers. A third approach involves the development of highly discriminatory quantitative methods for chromosome dosage analysis using digital polymerase chain reaction technology. It is likely that these and other methods yet to be developed would allow noninvasive prenatal diagnosis of chromosomal aneuploidies by maternal plasma nucleic acids to be realized in the near future.

Optimizing the yield and utility of circulating cell-free DNA from plasma and serum.
Xue X, Teare MD, Holen I, Zhu YM, Woll PJ.
Clin Chim Acta. 2009 404(2): 100-104

BACKGROUND: Cell-free DNA (CFDNA) in the plasma/serum of patients with cancer demonstrates tumour-associated genetic alterations, offering possibilities for diagnosis, prognostication and disease monitoring. There is wide variation in the reported levels of CFDNA, associated with different methods used to collect, process and analyze blood samples. We therefore evaluated different aspects of
laboratory protocols for the processing and purification of CFDNA in clinical studies.
METHODS: We evaluated and compared the QIAamp kit and a Triton/Heat/Phenol protocol (THP) for CFDNA purification. Total CFDNA was quantified by PicoGreen assay and SYBR-Green real-time PCR assay was used to amplify specific genes to estimate the efficiency of different protocols.
RESULTS: The efficiency of DNA extraction was 18.6% using the standard QIAamp protocol and 38.7% using the THP method (p < 0.0001, unpaired t-test). A modified QIAamp protocol that included a proteinase incubation stage and elution volumes up to 300 microl increased DNA yields, but was not as good as the THP method.
CONCLUSIONS: Blood samples should be kept at/or below room temperature (18 degrees C-22 degrees C) for no more than 2 h before plasma separation by double-spin. Because of its higher efficiency, low-cost and good-quality products, the THP protocol is preferred for extraction of CFDNA.

Optimized real-time quantitative PCR measurement of male fetal DNA in maternal plasma
Zimmermann BG, Holzgreve W, Avent N, Hahn S.
Laboratory for Prenatal Medicine, Department of Research/University Women's Hospital, University of Basel, Switzerland.
Ann N Y Acad Sci. 2006 Sep;1075: 347-349

DNA of fetal origin is present in the plasma of pregnant women. The quantitative measurement of circulatory fetal DNA (cfDNA) by real-time quantitative PCR (qPCR) has been applied to investigate a possible correlation between increased levels and pregnancy-related disorders. However, as the levels of cfDNA are close to the detection limit (LOD) of the method used, the measurements may not be reliable. This is also problematic for the evaluation of preanalytical steps, such as DNA extraction and cfDNA enrichment by size separation. We optimized a protocol for the qPCR analysis of the multi-copy sequence DYS14 on the Y chromosome. This was compared with an established assay for the single-copy SRY gene. Probit regression analysis showed that the limit of detection (LOD) of the DYS14 assay, (0.4 genome equivalents (GE)) and limit of quantification (LOQ) were 10-fold lower in comparison to SRY (4 GE). The levels of cfDNA obtained from the first trimester of pregnancy could be quantified with high precision by the DYS14 assay (CV below 25%) as opposed to the SRY measurements (26-140%). Additionally, fetal sex was correctly determined in all instances. The low copy numbers of fetal DNA in plasma of women in the first trimester of pregnancy can be measured reliably, targeting the DYS14 that is present in multiple copies per Y chromosome.

QIAamp Circulating Nucleic Acid Kit

For concentration and purification of free-circulating DNA and RNA from human plasma or serum
  • Concentration of nucleic acids, with high input and low elution volumes
  • Efficient recovery of fragmented DNA and RNA
  • No organic extraction or ethanol precipitation
  • Complete removal of contaminants and inhibitorsvolved in human development and pathology, are present in body fluids and represent new effective biomarkers.