Dataset Information

Improve sample preparation process for miRNA isolation from the culture cells by using silica fiber membrane.

ABSTRACT: In clinical applications of miRNAs, the purity and quality of the testing samples are very critical, especially the obtained tissue sample volume is limited. If the extracted miRNAs are contaminated or different in quality before analysis, it will increase the variance of the analysis result and make the medical information judgment incorrect and cannot be portable. Herein, we improved the commercially extraction kit by realizing the fundamental mechanism and hoped to serve finding optimal procedures for increasing the recovery of miRNAs extracted from cultured cells. In the adsorption process, the factors, like increasing the ethanol concentration or adding Ca²⁺, could influence the RNA adsorption were investigated. For the elution process, the effect caused by raising the elution temperature and raising the pH value of elution buffer was studied. Finally, the conditions for miRNA extraction are optimal modified by using a 65% (v/v) solution of ethanol in the adsorption process, and using TE buffer with the pH value of 8.0 and raising the temperature to 55 °C in the elution. According to the quantified results, the improved extraction kit can promote the recovery of endogenous miR-21 by about 6 times by using the optimal extraction conditions comparing with the miRNeasy Mini Kit.

SUBMITTER: Hu WP

PROVIDER: S-EPMC7713297 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Improve sample preparation process for miRNA isolation from the culture cells by using silica fiber membrane.

Hu Wen-Pin WP Chen Yu-Chi YC Chen Wen-Yih WY

Scientific reports 20201203 1

In clinical applications of miRNAs, the purity and quality of the testing samples are very critical, especially the obtained tissue sample volume is limited. If the extracted miRNAs are contaminated or different in quality before analysis, it will increase the variance of the analysis result and make the medical information judgment incorrect and cannot be portable. Herein, we improved the commercially extraction kit by realizing the fundamental mechanism and hoped to serve finding optimal proce ...[more]

PMID: 33273557

Similar Datasets

Project description:Bordetella pertussis, the aetiological agent of whooping cough, is re-emerging globally despite widespread vaccination. B. pertussis is highly infectious and, prior to vaccination programmes, was the leading cause of infant mortality. The WHO estimated that over 600?000 deaths are prevented annually by pertussis vaccination, but B. pertussis infection was still responsible for over 63?000 deaths globally in 2013. The re-emergence of B. pertussis has been linked to strains with inactive or absent major virulence factors included in vaccines such as pertactin, pertussis toxin and filamentous haemagglutinin. Thus, the molecular surveillance of currently circulating strains is critical in understanding and controlling B. pertussis. Such information provides data on strains to inform control measures and the identification of future vaccine antigens. Current surveillance and typing methods for B. pertussis rely on the availability of clinical isolates. However, since the 1990s, the majority of pertussis cases have been diagnosed by PCR, where an isolate is not needed. The rapid decline in the availability of B. pertussis isolates impacts our ability to monitor this infection. The growing uptake of next-generation sequencing (NGS) has offered the opportunity for culture-independent genome sequencing and typing of this fastidious pathogen. Therefore, the objective of the study was to optimize respiratory sample preparation, independent of culture, in order to type B. pertussis using NGS. The study compared commercial depletion kits and specimen-processing methods using selective lysis detergents. The goal was to deplete human DNA, the major obstacle for sequencing a pathogen directly from a clinical sample. Samples spiked with a clinically relevant amount of B. pertussis were used to provide comparison between the different methods. Commercial depletion kits including the MolYsis, Qiagen Microbiome and NEBNext Kits were tested. Previously published methods, for Saponin and TritonX-100, were also trialled as a depletion. The ratio of B. pertussis to human DNA was determined by real-time PCR for ERV3 and IS481 (as markers of human and B. pertussis DNA, respectively), then samples were sequenced using the Illumina NextSeq 500 platform. The number of human and B. pertussis sequenced reads were then compared between treatments. The results showed that commercial kits reduced the human DNA present, but also reduced the concentration of target B. pertussis. However, selective lysis with Saponin treatment resulted in almost undetectable levels of human DNA, with minimal loss of target B. pertussis DNA. Sequencing read depth improved five-fold in reads to B. pertussis. Our investigation delivered a potential protocol that will enable the public health laboratory surveillance of B. pertussis in the era of culture-independent testing.

Dataset Information

Improve sample preparation process for miRNA isolation from the culture cells by using silica fiber membrane.

Publications

Improve sample preparation process for miRNA isolation from the culture cells by using silica fiber membrane.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets