A new methodology to preserve the original proportion and integrity of cell‐free fetal DNA in maternal plasma during sample processing and storage

Fernando, M. Rohan; Chen, K.; Norton, Sheila E.; Krzyzanowski, Gary D.; Bourne, David W. A.; Hunsley, Bradford A.; Ryan, Wayne L.; Bassett, C. Andrew L.

doi:10.1002/pd.2484

Cited by 89 publications

(74 citation statements)

References 24 publications

(35 reference statements)

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“…The use high centrifugal force may destroy blood cells and release maternal DNA, subjecting it to the effects of DNases (Fernando et al, 2010). Thus, a two-step centrifugation process was used.…”

Section: Discussionmentioning

confidence: 99%

Isolation and analysis of cell-free fetal DNA from maternal peripheral blood in Chinese women

Yang¹,

Zhu²,

Qiu³

et al. 2015

Genet. Mol. Res.

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ABSTRACT. Non-invasive prenatal diagnosis is used to detect the genetic material of the fetus by isolating the cell-free fetal DNA (cffDNA) from maternal peripheral blood. In order to establish an isolation method for 18079 Isolation and analysis of cell free fetal DNA ©FUNPEC-RP www.funpecrp.com.br Genetics and Molecular Research 14 (4): 18078-18089 (2015) cffDNA from maternal peripheral blood in Chinese women, the cffDNA was acquired with a two-step centrifugation using a QlAamp DNA Blood mini kit. The SRY gene of plasma DNA was amplified by polymerase chain reaction (PCR). Real-time quantitative PCR was used to measure the concentration of cffDNA in maternal peripheral blood in different pregnant women. The results of the SRY gene amplification of plasma DNA from pregnant women was the same as that of the amniocyte DNA. The average concentration of cffDNA in maternal peripheral blood of pregnant women in different gestational stages was 0.98 ng/mL (0.26-1.49 ng/mL), 1.43 ng/mL (0.46-2.34 ng/mL), and 1.95 ng/mL (0.65-6.81 ng/mL) from early, middle, and late gestational stages, respectively. The mean of cffDNA from total DNA in plasma in different stages of gestation was 22.28% (9.86-27.81%). The lowest concentration of DNA amplified by nested-PCR in our research was 10 -4 -10 -3 ng/µL. The isolation method for cffDNA from maternal peripheral blood was successfully established and further research into its applications will be conducted.

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Section: Discussionmentioning

confidence: 99%

Isolation and analysis of cell-free fetal DNA from maternal peripheral blood in Chinese women

Yang¹,

Zhu²,

Qiu³

et al. 2015

Genet. Mol. Res.

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“…However, Fernando et al [34] demonstrated that cfDNA levels in blood samples drawn in cell-free DNA TM blood collection tubes were stable for up to 14 days at RT, while they decreased when blood was drawn in K3EDTA collection tubes. Barrett et al [28] showed that there was no significant difference for up to 3 days in the total cfDNA concentration when blood was drawn in cell-free DNA TM blood collection tubes, while it increased after 24 h when blood was drawn in K3EDTA collection tubes.…”

Section: Blood Collection Tubesmentioning

confidence: 95%

Pre-analytical Requirements for Analyzing Nucleic Acids from Blood

Messaoudi

Thierry

2014

Advances in Predictive, Preventive and Personalised Medicine

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Circulating nucleic acids have received an increasing scrutiny over the past decade with some applications, such as in prenatal diagnosis and oncology, being on the verge of use in clinical practice. It is crucial to implement optimal standardization of pre-analytical procedures. Currently, this domain has been poorly studied and there is no well-established procedure. This chapter examines the literature on the pre-analytical factors affecting nucleic acids from blood drawing to the storage of circulating cell-free DNA extracts ready for analysis and provides some elements as guidelines for a set procedure. In particular, this chapter reports on the choice between serum and plasma as the biological source but does not concern the actual nucleic acid extraction procedures (these will be dealt with in chapter "Circulating DNA and miRNA Isolation"). Currently, the lack of a standard operating procedure for the application of blood handling in a clinical setting is due to the lack of dispensing and sharing data among researchers as well as head-to-head comparative studies between techniques. This has led to in-house specific procedures that are, undoubtedly, prejudicial to the smooth translation of nucleic acid analysis into clinical practice. Hence, the proposed procedure should overcome this gap in technique.

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“…Recently cell-free DNA blood collection tubes (BCT) were introduced containing a stabilizing substance that prevents the extracellular DNA from being diluted by cellular DNA [267]. The use of these tubes might be a good choice when samples have to be shipped for processing to another laboratory but when the blood samples are processed in a reasonable time (several hours to 1 day) EDTA collection tubes are not inferior [268].…”

Section: Pre-analytic Variablesmentioning

confidence: 99%

Extracellular Nucleic Acids and Cancer

Fleischhacker¹,

Schmidt²

2014

Advances in Predictive, Preventive and Personalised Medicine

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Since the clear proof of the presence of tumor-associated genetic alterations in extracellular nucleic acids almost 20 years ago this field gained has attracted much interest. According to our current knowledge it seems as if all tumor-associated alterations found in tumor cells are also found in the extracellular environment. The isolation of extracellular nucleic acids from tumor patients and its genetic characterization with very sensitive and highly specific methods led to the concept of "liquid biopsy". This means that for follow-up analysis of tumor patients, the physicians no longer depend exclusively on a single examination of tissue biopsies (usually at the time of diagnosis) but are able by longitudinally analyzing the extracellular nucleic acids to follow the reaction of a tumor to e.g. a given therapy, the development of resistance mechanisms. In this chapter we will discuss the detection and characterization of different genetic (e.g. mutation analysis and structural variations as seen in microsatellites), epigenetic (e.g. hypermethylation of selected sequences) and regulatory alterations (as in different miRNA expression patterns found in tumor patients). We will also touch on some confounding factors that have to be taken into consideration as well as the functional and biological aspects of extracellular nucleic acids.

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A new methodology to preserve the original proportion and integrity of cell‐free fetal DNA in maternal plasma during sample processing and storage

Cited by 89 publications

References 24 publications

Isolation and analysis of cell-free fetal DNA from maternal peripheral blood in Chinese women

Isolation and analysis of cell-free fetal DNA from maternal peripheral blood in Chinese women

Pre-analytical Requirements for Analyzing Nucleic Acids from Blood

Extracellular Nucleic Acids and Cancer

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