Optimization of influencing factors of nucleic acid adsorption onto silica-coated magnetic particles: Application to viral nucleic acid extraction from serum

Sun, Ningning; Deng, Chao; Liu, Yi; Zhao, Xiaoli; Tang, Yan; Liu, Renxiao; Xia, Qiang; Yan, Wenlong; Ge, Guanglu

doi:10.1016/j.chroma.2013.11.059

Cited by 44 publications

(51 citation statements)

References 41 publications

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“…This buffer has a mid-range pKa, minimal salt effects, and binds only weakly to Ca, Mg, and Mn; it might wash out less RNA than 70% ethanol. Sun and coworkers 7 reported a decrease in RNA yield in their isolation protocol after washing with 70% ethanol. They also experienced that the initial binding of nucleic acids to silica beads did not occur in 70% ethanol without the addition of GuSCN.…”

Section: Resultsmentioning

confidence: 99%

“…The lysis buffer and Wash Buffer 1 contain the chaotropic agent guanidinium thiocyanate (GuSCN), which assists in the binding of the nucleic acids binding to silica particles and inhibits RNases. 3,7 Thus, Wash Buffer 1 would be less likely to result in loss of nucleic acids. As the concentration of the chaotropic agent is reduced throughout the multiple washing steps, nucleic acids will bind less strongly to the silica beads.…”

Section: Resultsmentioning

confidence: 99%

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Washed Away; How Not to Lose Your RNA during Isolation

Hønsvall

Robertson

2017

J Biomol Tech

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Cryptosporidium oocysts have extremely robust walls that protect the parasite against environmental pressures. Analyses must be sensitive to detect the few organisms (if any) present in environmental samples. After a series of negative nucleic acid amplification results on spiked samples, following a standard RNA isolation protocol, it seemed probable that oocyst RNA had been lost in the washing steps of the isolation protocol. By reducing both the volume of wash buffer and the number of washing steps, positive results could be re-established. Insufficient washing, however, seemed to prevent downstream analysis, probably because of inhibitory substances remaining in the RNA isolate. Nucleic acid isolation protocols for low numbers of "difficult" organisms should be adapted, according to the material to optimize the balance between removal of inhibitors and retention of target, thereby improving the performance of the technique.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Washed Away; How Not to Lose Your RNA during Isolation

Hønsvall

Robertson

2017

J Biomol Tech

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show abstract

“…Recently, silica or cellulose coated magnetic beads are applied to capturing either DNA or RNA in acidic 4M guanidinium thiocyanate [4,9,10]. This method is used for quick isolation of nucleic acid within 30min in the purpose of detection of viral infection [9].…”

Section: Introductionmentioning

confidence: 99%

“…This method is used for quick isolation of nucleic acid within 30min in the purpose of detection of viral infection [9]. In addition, magnetic oligo (dT) bead is applied to purify RNA species having poly(A) tail of mRNAs.…”

Section: Introductionmentioning

confidence: 99%

mirRICH, a simple method to enrich the small RNA fraction from over-dried RNA pellets

et al. 2018

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Techniques to isolate the small RNA fraction (<200nt) by column-based methods are commercially available. However, their use is limited because of the relatively high cost. We found that large RNA molecules, including mRNAs and rRNAs, are aggregated together in the presence of salts when RNA pellets are over-dried. Moreover, once RNA pellets are over-dried, large RNA molecules are barely soluble again during the elution process, whereas small RNA molecules (<100nt) can be eluted. We therefore modified the acid guanidinium thiocyanate-phenol-chloroform (AGPC)-based RNA extraction protocol by skipping the 70% ethanol washing step and over-drying the RNA pellet for 1 hour at room temperature. We named this novel small RNA isolation method “mirRICH.” The quality of the small RNA sequences was validated by electrophoresis, next-generation sequencing, and quantitative PCR, and the findings support that our newly developed column-free method can successfully and efficiently isolate small RNAs from over-dried RNA pellets.

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“…The effects of the inhibitors can be reduced by the selection of an appropriate nucleic acid isolation method. Numerous solid phase systems involving different micro-and nano-sized magnetic carriers and appropriate buffer systems have been used for DNA isolation [12,13]. The presence of interfering compounds released from magnetic particles decreased the PCR sensitivity or led to false-negative results [14].…”

Section: Introductionmentioning

confidence: 99%

The Evaluation of Magnetic Polymethacrylate-based Microspheres Used for Solid Phase DNA Micro-Extraction

et al. 2015

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Using magnetic particles as a solid-phase extraction system is the most frequently used micro-technique for DNA isolation. Particles with a complete covering of magnetic cores by a polymer are hence preferred. Quantitative polymerase chain reaction (qPCR) was used for the evaluation of the polymer coating efficiency of hydrophilic magnetic poly(2-hydroxyethyl methacrylate-co-glycidyl methacrylate) (P(HEMA-co-GMA)) and poly(glycidyl methacrylate) (PGMA) microspheres with/without carboxyl groups. The inhibition effect of magnetic microspheres was identified by the shift in Cq values (ΔCq) after the addition of different amounts of microspheres to PCR mixtures. With the increase of microsphere concentrations, the shift in Cq values to higher values was usually observed. P(HEMA-co-GMA) microspheres containing carboxyl groups extinguished the fluorescence at concentrations over 2 mg mL −1 in a PCR mixture without any influence on the synthesis of PCR products. No PCR products (inhibition of DNA amplification) were detected in the presence of more than 0.8 mg mL −1 in the PCR mixture of PGMA microspheres. Atomic force microscopy (AFM) was used for the determination of the surface morphology of the microspheres. The microspheres were spherical, and their surface was non-porous. OPEN ACCESSChromatography 2015, 2 157

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Optimization of influencing factors of nucleic acid adsorption onto silica-coated magnetic particles: Application to viral nucleic acid extraction from serum

Cited by 44 publications

References 41 publications

Washed Away; How Not to Lose Your RNA during Isolation

Washed Away; How Not to Lose Your RNA during Isolation

mirRICH, a simple method to enrich the small RNA fraction from over-dried RNA pellets

The Evaluation of Magnetic Polymethacrylate-based Microspheres Used for Solid Phase DNA Micro-Extraction

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