DNA/RNA Preparation for Molecular Detection

Thatcher, Stephanie A.

doi:10.1373/clinchem.2014.221374

Cited by 106 publications

(67 citation statements)

References 72 publications

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“…Fully automated NAE protocols have been developed for such equipment, using either solid-phase or magnetic beads methods [79]. However, high sample processivity is a positive aspect of automation while maintaining the sensitivity can be compromised, as low-copy NA targets might be lost [86]. Small versions of these robots are available and could be useful in laboratory settings with minimal infrastructure.…”

Section: Devices Used In Extraction Methodsmentioning

confidence: 99%

Current Nucleic Acid Extraction Methods and Their Implications to Point-of-Care Diagnostics

Ali

Rampazzo

Costa

et al. 2017

BioMed Research International

241

237

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Nucleic acid extraction (NAE) plays a vital role in molecular biology as the primary step for many downstream applications. Many modifications have been introduced to the original 1869 method. Modern processes are categorized into chemical or mechanical, each with peculiarities that influence their use, especially in point-of-care diagnostics (POC-Dx). POC-Dx is a new approach aiming to replace sophisticated analytical machinery with microanalytical systems, able to be used near the patient, at the point of care or point of need. Although notable efforts have been made, a simple and effective extraction method is still a major challenge for widespread use of POC-Dx. In this review, we dissected the working principle of each of the most common NAE methods, overviewing their advantages and disadvantages, as well their potential for integration in POC-Dx systems. At present, it seems difficult, if not impossible, to establish a procedure which can be universally applied to POC-Dx. We also discuss the effects of the NAE chemicals upon the main plastic polymers used to mass produce POC-Dx systems. We end our review discussing the limitations and challenges that should guide the quest for an efficient extraction method that can be integrated in a POC-Dx system.

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Section: Devices Used In Extraction Methodsmentioning

confidence: 99%

Current Nucleic Acid Extraction Methods and Their Implications to Point-of-Care Diagnostics

Ali

Rampazzo

Costa

et al. 2017

BioMed Research International

241

237

View full text Add to dashboard Cite

show abstract

“…Further information about sample preparation techniques and some commercial kits available can be found in Ref. [52]. Different commercial kits demonstrated satisfactory RNA yield, but differences in the quality of extracted RNA were observed, which can interfere on the downstream analysis [53].…”

Section: Rna Isolation and Extractionmentioning

confidence: 99%

RNA‐seq: Applications and Best Practices

Pereira¹,

Imada²,

MunizGuedes³

2017

Applications of RNA-Seq and Omics Strategies - From Microorganisms to Human Health

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RNA-sequencing (RNA-seq) is the state-of-the-art technique for transcriptome analysis that takes advantage of high-throughput next-generation sequencing. Although being a powerful approach, RNA-seq imposes major challenges throughout its steps with numerous caveats. There are currently many experimental options available, and a complete comprehension of each step is critical to make right decisions and avoid getting into inconclusive results. A complete workflow consists of: (1) experimental design; (2) sample and library preparation; (3) sequencing; and (4) data analysis. RNA-seq enables a wide range of applications such as the discovery of novel genes, gene/transcript quantification, and differential expression and functional analysis. This chapter will encompass the main aspects from sample preparation to downstream data analysis. It will be discussed how to obtain high-quality samples, replicates amount, library preparation, sequencing platforms and coverage, focusing on best recommended practices based on specialized literature. Basic techniques and well-known algorithms are presented and discussed, guiding both beginners and experienced users in the implementation of reliable experiments.Sanger sequencing [6], but with advances in next-generation sequencing technology (NGS), transcriptomic studies have evolved considerably and RNA-seq [7,8] became the state-of-art for transcriptome analysis.RNA-seq consists of the direct sequencing of transcripts by NGS. Several NGS platforms [9][10][11] are commercially available nowadays. In general, an RNA set of interest is converted to a library of complementary DNA (cDNA) fragments and sequenced in a high-throughput manner. Compared to ESTs, RNA-seq provides better resolution and representativeness, whereas when compared to microarrays, the independence of reference sequences facilitates the discovery of novel genes and isoforms [8].RNA-seq experiments harbors challenges from the experimental design to data analysis. Since a complete comprehension of each step is critical to make right decision, this chapter will encompass essential principles required for a successful RNA-seq experiment, focusing on best recommended practices based on specialized and recent literature. Basic techniques and well-known algorithms are presented and discussed, guiding both beginners and experienced users in the implementation of reliable experiments. Experimental designIn order to obtain a successful RNA-seq experiment, it is critical to have a good experimental design. Despite its importance, a proper planning is not always done. There are many experimental options available, and to fully comprehend each step, it is essential to make right decisions, avoiding inconclusive results. These choices depend on extrinsic (e.g., cost, time, samples availability) and intrinsic (e.g., experimental design complexity, transcriptional variability among tissues, samples and organisms) factors. The amount of available resources is usually the main extrinsic limiting factor driving researchers' decisio...

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“…There is no single method for every application; many factors, including the target viral pathogen(s), sample volume, final volume needed for testing, yield, and upstream concentration, should be considered when a new sample preparation system is introduced into clinical laboratories [67]. In addition, various sample types have been used for detecting CMV and EBV DNA, with no consensus regarding the optimal sample type [38910].…”

Section: Introductionmentioning

confidence: 99%

Automated Nucleic Acid Extraction Systems for Detecting Cytomegalovirus and Epstein-Barr Virus Using Real-Time PCR: A Comparison Study Between the QIAsymphony RGQ and QIAcube Systems

et al. 2017

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Background: Cytomegalovirus (CMV) and Epstein-Barr virus (EBV) are increasingly important in immunocompromised patients. Nucleic acid extraction methods could affect the results of viral nucleic acid amplification tests. We compared two automated nucleic acid extraction systems for detecting CMV and EBV using real-time PCR assays. Methods:One hundred and fifty-three whole blood (WB) samples were tested for CMV detection, and 117 WB samples were tested for EBV detection. Viral nucleic acid was extracted in parallel by using QIAsymphony RGQ and QIAcube (Qiagen GmbH, Germany), and real-time PCR assays for CMV and EBV were performed with a Rotor-Gene Q real-time PCR cycler (Qiagen). Detection rates for CMV and EBV were compared, and agreements between the two systems were analyzed. Conclusions: Automated nucleic acid extraction systems have different performances and significantly affect the detection of viral pathogens. The QIAsymphony RGQ system appears to be superior to the QIAcube system for detecting CMV and EBV. A suitable sample preparation system should be considered for optimized nucleic acid amplification in clinical laboratories.

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DNA/RNA Preparation for Molecular Detection

Cited by 106 publications

References 72 publications

Current Nucleic Acid Extraction Methods and Their Implications to Point-of-Care Diagnostics

Current Nucleic Acid Extraction Methods and Their Implications to Point-of-Care Diagnostics

RNA‐seq: Applications and Best Practices

Automated Nucleic Acid Extraction Systems for Detecting Cytomegalovirus and Epstein-Barr Virus Using Real-Time PCR: A Comparison Study Between the QIAsymphony RGQ and QIAcube Systems

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