Carrier RNA enhancement of recovery of DNA from dilute solutions

Gallagher, Margaret; Burke, William F.; Orzech, Karen A.

doi:10.1016/s0006-291x(87)80506-5

Cited by 16 publications

(11 citation statements)

References 1 publication

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“…Differences in the concentrations of the components used in these experiments likely account for this discrepancy. The significant positive effect of cations and cells with the Liat assay were similar in magnitude to SRS and suggested improved extraction efficiency by a carrier effect from cellular nucleic acid, especially with low viral RNA copy numbers [13,14]. Pooled NCS was shown to have a similar but less pronounced effect.…”

Section: Discussionmentioning

confidence: 98%

Simulated Respiratory Secretion for Use in the Development of Influenza Diagnostic Assays

Bose¹,

McCaul²,

Mei³

et al. 2016

PLoS ONE

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Many assays have been developed for the detection of influenza virus which is an important respiratory pathogen. Development of these assays commonly involves the use of human clinical samples for validation of their performance. However, clinical samples can be difficult to obtain, deteriorate over time, and be inconsistent in composition. The goal of this study was to develop a simulated respiratory secretion (SRS) that could act as a surrogate for clinical samples. To this end, we determined the effects major respiratory secretion components (Na+, K+, Ca2+, cells, albumin IgG, IgM, and mucin) have on the performance of influenza assays including both nucleic acid amplification and rapid antigen assays. Minimal effects on the molecular assays were observed for all of the components tested, except for serum derived human IgG, which suppressed the signal of the rapid antigen assays. Using dot blots we were able to show anti-influenza nucleoprotein IgG antibodies are common in human respiratory samples. We composed a SRS that contained mid-point levels of human respiratory sample components and studied its effect compared to phosphate buffered saline and virus negative clinical sample matrix on the Veritor, Sofia, CDC RT-PCR, Simplexa, cobas Liat, and Alere i influenza assays. Our results demonstrated that a SRS can interact with a variety of test methods in a similar manner to clinical samples with a similar impact on test performance.

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Section: Discussionmentioning

confidence: 98%

Simulated Respiratory Secretion for Use in the Development of Influenza Diagnostic Assays

Bose¹,

McCaul²,

Mei³

et al. 2016

PLoS ONE

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“…The rationale for this inclusion is that while silica exhibits a relatively high affinity for DNA, recovery can sometimes prove problematic in low DNA solutions such as found circulating within the bloodstream. The use of carrier RNA helps facilitate the precipitation of DNA so that it can be more readily captured onto the silica surface of the SSB, resulting in significantly higher yields [23]. …”

Section: Discussionmentioning

confidence: 99%

Extraction and processing of circulating DNA from large sample volumes using methylation on beads for the detection of rare epigenetic events

Keeley

Stark

Pisanic

et al. 2013

Clinica Chimica Acta

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The use of methylated tumor-specific circulating DNA has shown great promise as a potential cancer biomarker. Nonetheless, the relative scarcity of tumor-specific circulating DNA presents a challenge for traditional DNA extraction and processing techniques. Here we demonstrate a single tube extraction and processing technique dubbed “methylation on beads” that allows for DNA extraction and bisulfite conversion for up to 2 ml of plasma or serum. In comparison to traditional techniques including phenol chloroform and alcohol extraction, methylation on beads yields a 1.5 to 5-fold improvement in extraction efficiency. The technique results in far less carryover of PCR inhibitors yielding analytical sensitivity improvements of over 25-fold. The combination of improved recovery and sensitivity make possible the detection of rare epigenetic events and the development of high sensitivity epigenetic diagnostic assays.

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“…Lyophilized poly-A RNA (approx. 20 ng per sample) is added to the sample to improve NA isolation yield [90, 91, 92]. This may be beneficial due to one or a combination of several effects: (1) the carrier RNA acts as a sacrificial substrate for RNAases that would otherwise degrade the template; (2) the carrier RNA enhances precipitation and binding of NA to the solid-phase membrane, and (3) small amounts of NA are strongly and irreversibly adsorbed on the NA binding phase and cannot be desorbed to serve as templates for amplification.…”

Section: Notesmentioning

confidence: 99%

Microfluidic Devices for Nucleic Acid (NA) Isolation, Isothermal NA Amplification, and Real-Time Detection

Mauk

Liu

Sadik

et al. 2014

Methods in Molecular Biology

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Molecular (nucleic acid)-based diagnostics tests have many advantages over immunoassays, particularly with regard to sensitivity and specificity. Most on-site diagnostic tests, however, are immunoassay-based because conventional nucleic acid-based tests (NATs) require extensive sample processing, trained operators, and specialized equipment. To make NATs more convenient, especially for point-of-care diagnostics and on-site testing, a simple plastic microfluidic cassette ("chip") has been developed for nucleic acid-based testing of blood, other clinical specimens, food, water, and environmental samples. The chip combines nucleic acid isolation by solid-phase extraction; isothermal enzymatic amplification such as LAMP (Loop-mediated AMPlification), NASBA (Nucleic Acid Sequence Based Amplification), and RPA (Recombinase Polymerase Amplification); and real-time optical detection of DNA or RNA analytes. The microfluidic cassette incorporates an embedded nucleic acid binding membrane in the amplification reaction chamber. Target nucleic acids extracted from a lysate are captured on the membrane and amplified at a constant incubation temperature. The amplification product, labeled with a fluorophore reporter, is excited with a LED light source and monitored in situ in real time with a photodiode or a CCD detector (such as available in a smartphone). For blood analysis, a companion filtration device that separates plasma from whole blood to provide cell-free samples for virus and bacterial lysis and nucleic acid testing in the microfluidic chip has also been developed. For HIV virus detection in blood, the microfluidic NAT chip achieves a sensitivity and specificity that are nearly comparable to conventional benchtop protocols using spin columns and thermal cyclers.

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Carrier RNA enhancement of recovery of DNA from dilute solutions

Cited by 16 publications

References 1 publication

Simulated Respiratory Secretion for Use in the Development of Influenza Diagnostic Assays

Simulated Respiratory Secretion for Use in the Development of Influenza Diagnostic Assays

Extraction and processing of circulating DNA from large sample volumes using methylation on beads for the detection of rare epigenetic events

Microfluidic Devices for Nucleic Acid (NA) Isolation, Isothermal NA Amplification, and Real-Time Detection

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