Desalting DNA by Drop Dialysis Increases Library Size upon Transformation

Saraswat, Mayank; Grand, Ralph S.; Patrick, Wayne M.

doi:10.1271/bbb.120767

Cited by 7 publications

(4 citation statements)

References 11 publications

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“…In addition, it was necessary to desalt the extracts because the retention of the miRNAs was compromised as a result of the high abundance of salts. Drop dialysis was performed because it was a simple, rapid, and highly effective method to remove the salts from the small volume samples. , …”

Section: Resultsmentioning

confidence: 99%

“…Drop dialysis was performed because it was a simple, rapid, and highly effective method to remove the salts from the small volume samples 47,48 .…”

Section: Microrna Detection In Cancer Serummentioning

confidence: 99%

“…Drop dialysis was performed because it was a simple, rapid, and highly effective method to remove the salts from the small volume samples. 47,48 The sample pretreatments were optimized with healthy control serum samples spiked with the standard miRNAs, miR-21-5p and let-7g-5p. The PCA-extraction procedure was selected because of the limited compatibility of the TRIzol Reagent with MS, due to the presence of high concentrations of chaotropic salts (i.e., guanidinium thiocyanate).…”

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confidence: 99%

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Analysis of Circulating microRNAs and Their Post-Transcriptional Modifications in Cancer Serum by On-Line Solid-Phase Extraction–Capillary Electrophoresis–Mass Spectrometry

et al. 2018

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In this paper, an on-line solid-phase extraction capillary electrophoresis-mass spectrometry (SPE-CE-MS) method is described for the purification, preconcentration, separation, and characterization of endogenous microRNA (miRNA) and their post-transcriptional modifications in serum. First, analysis by CE-MS was optimized using a standard mixture of hsa-miR-21-5p (miR-21-5p) and hsa-let-7g-5p (let-7g-5p). For SPE-CE-MS, a commercial silicon carbide (SiC) resin was used to prepare the microcartridges. Under the optimized conditions with standards, the microcartridge lifetime (>25 analyses) and repeatability (2.8% RSD for the migration times; 4.4 and 6.4% RSD for the miR-21-5p and let-7g-5p peak areas, respectively) were good, the method was linear between 25 and 100 nmol·L, and the limit of detection (LOD) was around 10 nmol·L (50 times lower than by CE-MS). In order to analyze human serum samples, an off-line sample pretreatment based on phenol/chloroform/isoamyl alcohol (PCA) extraction was necessary prior to SPE-CE-MS. The potential of the SPE-CE-MS method to screen for B-cell chronic lymphocytic leukemia (CLL) was demonstrated by an analysis of serum samples from healthy controls and patients. MicroRNAs, specifically miR-21-5p and a 23 nucleotide long 5'-phosphorylated miRNA with 3'-uridylation (iso-miR-16-5p), were only detected in the CLL patients.

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Section: Resultsmentioning

confidence: 99%

“…Drop dialysis was performed because it was a simple, rapid, and highly effective method to remove the salts from the small volume samples 47,48 .…”

Section: Microrna Detection In Cancer Serummentioning

confidence: 99%

mentioning

confidence: 99%

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Analysis of Circulating microRNAs and Their Post-Transcriptional Modifications in Cancer Serum by On-Line Solid-Phase Extraction–Capillary Electrophoresis–Mass Spectrometry

et al. 2018

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“…DNA for digital PCR was prepared (not purified) directly from living cells attached on the culturing surface of a 96-plate using a simple, quick and inexpensive method which includes an initial cell lysis by HotShot 6 and subsequent desalting by Drop-Dialysis 7 .…”

Section: Dna Preparationmentioning

confidence: 99%

A novel genetic- and cell-based tool for assessing the efficacy and toxicity of anticancer drugs in vitro

Jiang¹,

Alster²,

Hanken³

2016

BIOMED PAP

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Aims.To develop an in vitro tool for assessing the efficacy and toxicity of anticancer drugs using mixed culture containing both tumor and non-tumor cells. Such in vitro tool should have high application potential in drug-screening and personalized cancer care. Methods. Fibroblasts were spiked as non-tumor cells into tumor cells of an established line. The mixed culture was treated with a test drug at various concentrations. After the treatment, DNA was prepared directly from the survived adhesive cells in the wells of the 96-well plates using a simple and inexpensive method, and subjected to digital PCR for measuring relative copy numbers of a target gene NF1 to that of a reference gene RPP30. The NF1 gene is known to be heterozygously deleted in these tumor cells while the RPP30 gene has two copies in both tumor and non-tumor cells. Using the NF1/ RPP30 ratios resulting from the dual digital PCR assay, the proportions of tumor cells were calculated for each drug concentration. Results. Digital PCR confirmed that the tumor cells have only one copy of the NF1 gene while the non-tumor fibroblasts have two copies. By contrast, both types of cells have two copies of the reference gene RPP30. Using the ratio of the two genes, we successfully calculated the proportion of tumor cells which decreased as the dose of the test drug increased up to a certain concentration, indicating that the drug is more effective for the tumor cells than for the non-tumor cells in this dose-range. At the highest dose, we observed a slight increase in the proportion of tumor cells, likely reflecting the toxic effect of the drug on both tumor and non-tumor cells.Conclusion. This pilot study demonstrated the feasibility of a genetic-and cell-based tool for testing efficacy and toxicity of anticancer drugs in vitro. The promising results suggest that additional efforts are merited, for further development since such a tool will likely have high application potential (1) in drug discovery where it enables simultaneously assessing therapeutic effect on target cells and toxic effect on non-target cells, and (2) in personalized adjuvant chemotherapy where multiple drugs can be tested in primary cultures derived from surgically removed tumor.

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Error-Prone PCR and Effective Generation of Gene Variant Libraries for Directed Evolution

Copp

Hanson‐Manful

Ackerley

et al. 2014

Methods in Molecular Biology

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Any single-enzyme directed evolution strategy has two fundamental requirements: the need to efficiently introduce variation into a gene of interest and the need to create an effective library from those variants. Generation of a maximally diverse gene library is particularly important when employing nontargeted mutagenesis strategies such as error-prone PCR (epPCR), which seek to explore very large areas of sequence space. Here we present comprehensive protocols and tips for using epPCR to generate gene variants that exhibit a relatively balanced spectrum of mutations and for capturing as much diversity as possible through effective cloning of those variants. The detailed library preparation methods that we describe are generally applicable to any directed evolution strategy that uses restriction enzymes to clone gene variants into an expression plasmid.

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Desalting DNA by Drop Dialysis Increases Library Size upon Transformation

Cited by 7 publications

References 11 publications

Analysis of Circulating microRNAs and Their Post-Transcriptional Modifications in Cancer Serum by On-Line Solid-Phase Extraction–Capillary Electrophoresis–Mass Spectrometry

Analysis of Circulating microRNAs and Their Post-Transcriptional Modifications in Cancer Serum by On-Line Solid-Phase Extraction–Capillary Electrophoresis–Mass Spectrometry

A novel genetic- and cell-based tool for assessing the efficacy and toxicity of anticancer drugs in vitro

Error-Prone PCR and Effective Generation of Gene Variant Libraries for Directed Evolution

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