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
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
The presence of DNA and RNA in plasma of patients has been recognised
since the 1970s - see table.
does the free CNA come from - The nucleic acid sources:
There are several
possible sources of blood DNA,
- break down of blood cells
- brake down of any pathogens, e.g. bacteria or
- leucocyte surface DNA
- spontaneous release of a newly synthesised DNA
- spontaneous release of DNA/RNA-lipoprotein
from healthy cells
Only small amounts
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.
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.
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 is only
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.
are evolutionarily conserved,
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
- CNA as biomarkers in
various cancer types
A range of markers
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
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.
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 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,
Front. Genet., 19 April 2013
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.
Nucleic Acids in
Plasma and Serum - An Overview
Y. M. DENNIS LO
Annals of the New York Academy of Sciences 2001
Volume 945 Issue CIRCULATING NUCLEIC ACIDS IN PLASMA OR SERUM II, Pages
1 - 7
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.
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
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.
(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
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.
developments involving cell-free (circulating) nucleic acids.
Tong YK, Lo YM.
Clin Chim Acta. 2006 363(1-2): 187-196
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
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.
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
APPROACH: We reviewed published literature up to 2002 on the topic,
with a particular emphasis on reports published between 1996 and 2002.
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.
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.
resolves the asymmetry of siRNA strand degradation in human blood serum.
Hoerter JA, Walter NG
RNA. 2007 13(11): 1887-1893.
(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.
and RNA markers in blood: a tool for early detection, diagnostics, and
Bremnes RM, Sirera R, Camps C.
Lung Cancer 2005 49(1): 1-12. Review.
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.
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.
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.
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.
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
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.
nucleic acids in plasma or
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
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).
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)....................
nucleic acids in
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.
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
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
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
|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. firstname.lastname@example.org
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.
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
low elution volumes
- Efficient recovery of fragmented DNA and RNA
- No organic extraction or ethanol precipitation
- Complete removal of contaminants and
in human development and pathology, are present in body fluids and
represent new effective biomarkers.