Comparison of Protocols for Extracting Circulating DNA and RNA from Maternal Plasma

Chiu, Rossa W.K.; Lui, Wing Bong; El-Sheikhah, Ahmad; Chan, Anthony T.C.; Lau, Tze Kin; Nicolaides, Kypros H.; Lo, Y.M. Dennis

doi:10.1373/clinchem.2005.056366

Cited by 13 publications

(9 citation statements)

References 7 publications

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“…Plasma DNAs were extracted using QIAmp DNA Blood Mini kit (Qiagen), as previously described [19], [20] with modifications. In brief, 600 µl of plasma was used and DNA was eluted with 40 µl of DNase-free water.…”

Section: Methodsmentioning

confidence: 99%

Quantitative Analysis and Diagnostic Significance of Methylated SLC19A3 DNA in the Plasma of Breast and Gastric Cancer Patients

Leung

Shin

et al. 2011

PLoS ONE

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BackgroundPreviously, we have examined the methylation status of SLC19A3 (solute carrier family 19, member 3) promoter and found that SLC19A3 was epigenetically down-regulated in gastric cancer. Here, we aim to develop a new biomarker for cancer diagnosis using methylated SLC19A3 DNA in plasma.Methodology/Principal FindingsSLC19A3 gene expression was examined by RT-qPCR. Methylation status of SLC19A3 promoter was evaluated by methylation-specific qPCR. SLC19A3 expression was significantly down-regulated in 80% (12/15) of breast tumors (P<0.005). Breast tumors had significant increase in methylation percentage when compared to adjacent non-tumor tissues (P<0.005). A robust and simple methylation-sensitive restriction enzyme digestion and real-time quantitative PCR (MSRED-qPCR) was developed to quantify SLC19A3 DNA methylation in plasma. We validated this biomarker in an independent validation cohort of 165 case-control plasma including 60 breast cancer, 45 gastric cancer patients and 60 healthy subjects. Plasma SLC19A3 methylated DNA level was effective in differentiating both breast and gastric cancer from healthy subjects. We further validated this biomarker in another independent blinded cohort of 78 plasma including 38 breast cancer, 20 gastric cancer patients and 20 healthy subjects. The positive predictive values for breast and gastric cancer were 90% and 85%, respectively. The negative predictive value of this biomarker was 85%. Elevated level in plasma has been detected not only in advanced stages but also early stages of tumors. The positive predictive value for ductal carcinoma in situ (DCIS) cases was 100%.ConclusionsThese results suggested that aberrant SLC19A3 promoter hypermethylation in plasma may be a novel biomarker for breast and gastric cancer diagnosis.

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Section: Methodsmentioning

confidence: 99%

Quantitative Analysis and Diagnostic Significance of Methylated SLC19A3 DNA in the Plasma of Breast and Gastric Cancer Patients

Leung

Shin

et al. 2011

PLoS ONE

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“…Since fetal CNAs exist not only as DNA but also RNA, different extraction methods may be required for the individual class of CNA molecules 56 57. For example, a column-based extraction method has been shown to recover fetal DNA at a higher concentration while a magnetic-bead based protocol has been shown to benefit fetal RNA extraction 56.…”

Section: Preanalytical Issues In Fetal Cna Detectionmentioning

confidence: 99%

“…For example, a column-based extraction method has been shown to recover fetal DNA at a higher concentration while a magnetic-bead based protocol has been shown to benefit fetal RNA extraction 56. Standardisation of these extraction protocols is clearly needed to give comparable results across clinical laboratories.…”

Section: Preanalytical Issues In Fetal Cna Detectionmentioning

confidence: 99%

Detection of circulating fetal nucleic acids: a review of methods and applications

Hung

Chiu

2009

J Clin Pathol

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The discovery of cell-free circulating fetal nucleic acids in maternal plasma has opened up new possibilities in non-invasive prenatal diagnosis. The rapid advancement of this field in the past decade is catalysed by the discovery of new classes of fetal nucleic acid markers and technological developments in nucleic acid detection and amplification. In this review, some of the more significant recent developments in this field will be discussed, including the detection of single molecule, chromosomal aneuploidies, single nucleotide variations and placental microRNAs in maternal plasma.

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“…These applications can be divided into two groups: those that require the qualitative analysis of circulating fetal DNA, and those that require its quantitative assessment. These analyses typically start with the separation of plasma from the cellular components of maternal blood, followed by the extraction of DNA from the plasma fraction 19,20 . For both of these types of application, it is important to bear in mind that fetal DNA only represents a minor fraction of the DNA that is present in maternal plasma 5 .…”

Section: Clinical Applicationsmentioning

confidence: 99%

Prenatal diagnosis: progress through plasma nucleic acids

Chiu

2006

Nat Rev Genet

164

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Comparison of Protocols for Extracting Circulating DNA and RNA from Maternal Plasma

Cited by 13 publications

References 7 publications

Quantitative Analysis and Diagnostic Significance of Methylated SLC19A3 DNA in the Plasma of Breast and Gastric Cancer Patients

Quantitative Analysis and Diagnostic Significance of Methylated SLC19A3 DNA in the Plasma of Breast and Gastric Cancer Patients

Detection of circulating fetal nucleic acids: a review of methods and applications

Prenatal diagnosis: progress through plasma nucleic acids

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