Dimethyl adipimidate/Thin film Sample processing (DTS); A simple, low-cost and versatile nucleic acid extraction assay for downstream analysis

AbstractDespite advances in automated liquid handling and microfluidics, preparing samples for RNA sequencing at scale generally requires expensive equipment, which is beyond the reach of many academic labs. Manual sample preparation remains a slow, expensive, and error-prone process. Here, we describe a low-cost, semi-automated pipeline to extract cell-free RNA (cfRNA) that increases sample throughput by 12-fold while reducing time spent and cost by nearly 11-fold and 3-fold respectively. This pipeline is generalizable for many nucleic acid extraction applications, thereby increasing the scale of studies, which can be performed in small research labs.

Download Full-text

Advances in Fully Automated Nucleic Acid Extraction System Based on Magnetic Nanobeads

Nanoscience and Nanotechnology Letters ◽

10.1166/nnl.2019.3051 ◽

2019 ◽

Vol 11 (12) ◽

pp. 1633-1643

Author(s):

Zhu Chen ◽

Changhu Xiao ◽

Manling Tang ◽

Yuyue Xu ◽

Ziyu He ◽

...

Keyword(s):

Nucleic Acid ◽

High Throughput ◽

Point Of Care ◽

Low Cost ◽

Future Trend ◽

Acid Extraction ◽

Nucleic Acid Extraction ◽

Advantages And Disadvantages ◽

Good Repeatability ◽

Magnetic Nanobeads

Using magnetic nanobeads (MNBs) to extract nucleic acids is an efficient, inexpensive, easy automation, high throughput and good repeatability, instead of the traditional nucleic acid extraction (NAE) methods. Advances in fully automated MNBs-based nucleic acid extraction systems (MNAES) can push the frontiers of point-of-care testing (POCT) devices towards low-cost, automation, and enhanced accuracy molecular-level diagnostics. So, this paper introduces the pipettingbased MNAES with position of magnetic separation kit or tip, and based on magnetic bar MNAES with blending manner is shock or rotation. Further, advantages and disadvantages of various MNAES are compared. We envisage that the future trend in molecular diagnosis and monitoring will be facilitation, intelligent, miniaturization, and high throughput MNAES with sample-in-answer-out capability.

Download Full-text

Automated nucleic acid extraction from whole blood, B. subtilis, E. coli, and Rift Valley fever virus on a centrifugal microfluidic LabDisk

RSC Advances ◽

10.1039/c5ra03399c ◽

2015 ◽

Vol 5 (41) ◽

pp. 32144-32150 ◽

Cited By ~ 27

Author(s):

O. Strohmeier ◽

S. Keil ◽

B. Kanat ◽

P. Patel ◽

M. Niedrig ◽

...

Keyword(s):

Nucleic Acid ◽

Whole Blood ◽

Rift Valley ◽

Rift Valley Fever ◽

Rna Virus ◽

Rift Valley Fever Virus ◽

Low Cost ◽

Acid Extraction ◽

Nucleic Acid Extraction ◽

Valley Fever

We present total nucleic acid extraction from whole blood, Gram-positiveBacillus subtilis, Gram-negativeEscherichia coli, andRift Valley feverRNA virus on a low-cost, centrifugal microfluidic cartridge processed in a portable processing device.

Download Full-text

Optimizing nucleic acid extraction and transcriptome evaluation from low-input, fixed clinical samples

10.1101/2020.06.02.122150 ◽

2020 ◽

Author(s):

Katie M. Campbell ◽

Egmidio Medina ◽

Ignacio Baselga Carretero ◽

Yaroslav Teper ◽

Rangasamy Elumalai ◽

...

Keyword(s):

Nucleic Acid ◽

Rna Sequencing ◽

Clinical Samples ◽

Acid Extraction ◽

Nucleic Acid Extraction ◽

Sequencing Library ◽

Ffpe Samples ◽

Agilent Sureselect ◽

Downstream Analysis ◽

The Impact

AbstractTumor biopsies are commonly formalin-fixed and paraffin-embedded (FFPE) for long-term and efficient storage. However, FFPE preservation can greatly compromise the quality of samples, the extraction of nucleic acids, and feasibility of downstream studies, including RNA sequencing. These challenges are especially evident in the studies of clinical trial samples, where the sizes of biopsies often limit the amount of material available for study. Here, we evaluate two nucleic acid extraction kits (Covaris truXTRAC FFPE tNA Plus, QIAGEN miRNeasy FFPE) and three hybridized-capture-based RNA sequencing library preparations (Agilent SureSelect XT RNA Direct, Agilent SureSelect XT HS, and Illumina TruSeq RNA Exome) to evaluate the impact of these sample processing steps on transcriptome evaluation in a melanoma biopsy procured in a clinical setting and preserved by FFPE. While there exist many options for extraction and library preparation that are appropriate for RNA sequencing from FFPE samples, we observed that combinations of experimental approaches may have subtle impacts on downstream analysis, including gene expression quantification and fusion detection.

Download Full-text

Differing Effects of Standard and Harsh Nucleic Acid Extraction Procedures on Diagnostic Helminth Real-Time PCRs Applied to Human Stool Samples

Pathogens ◽

10.3390/pathogens10020188 ◽

2021 ◽

Vol 10 (2) ◽

pp. 188

Author(s):

Tanja Hoffmann ◽

Andreas Hahn ◽

Jaco J. Verweij ◽

Gérard Leboulle ◽

Olfert Landt ◽

...

Keyword(s):

Nucleic Acid ◽

Real Time ◽

Real Time Pcr ◽

Acid Extraction ◽

Nucleic Acid Extraction ◽

Pcr Assay ◽

Bead Beating ◽

Stool Samples ◽

Pcr Assays ◽

Human Stool

This study aimed to assess standard and harsher nucleic acid extraction schemes for diagnostic helminth real-time PCR approaches from stool samples. A standard procedure for nucleic acid extraction from stool and a procedure including bead-beating as well as proteinase K digestion were compared with group-, genus-, and species-specific real-time PCR assays targeting helminths and nonhelminth pathogens in human stool samples. From 25 different in-house and commercial helminth real-time PCR assays applied to 77 stool samples comprising 67 historic samples and 10 external quality assessment scheme samples positively tested for helminths, higher numbers of positive test results were observed after bead-beating-based nucleic acid extraction for 5/25 (20%) real-time PCR assays irrespective of specificity issues. Lower cycle threshold values were observed for one real-time PCR assay after the standard extraction scheme, and for four assays after the bead-beating-based scheme. Agreement between real-time PCR results after both nucleic acid extraction strategies according to Cohen’s kappa ranged from poor to almost perfect for the different assays. Varying agreement was observed in eight nonhelminth real-time PCR assays applied to 67 historic stool samples. The study indicates highly variable effects of harsh nucleic acid extraction approaches depending on the real-time PCR assay used.

Download Full-text

Forensic evaluation of two nucleic acid extraction systems and validation of a RT-qPCR protocol for identification of SARS-CoV-2 in post-mortem nasopharyngeal swabs

Forensic Science International ◽

10.1016/j.forsciint.2021.110775 ◽

2021 ◽

pp. 110775

Author(s):

Pedro A. Barrio ◽

Amparo Fernández-Rodríguez ◽

Pablo Martín ◽

Coro Fernández ◽

Lourdes Fernández ◽

...

Keyword(s):

Nucleic Acid ◽

Forensic Evaluation ◽

Acid Extraction ◽

Post Mortem ◽

Nucleic Acid Extraction

Download Full-text

Evaluation of an Automated High-Throughput Liquid-Based RNA Extraction Platform on Pooled Nasopharyngeal or Saliva Specimens for SARS-CoV-2 RT-PCR

Viruses ◽

10.3390/v13040615 ◽

2021 ◽

Vol 13 (4) ◽

pp. 615

Author(s):

Allen Wing-Ho Chu ◽

Cyril Chik-Yan Yip ◽

Wan-Mui Chan ◽

Anthony Chin-Ki Ng ◽

Dream Lok-Sze Chan ◽

...

Keyword(s):

Nucleic Acid ◽

High Throughput ◽

Rna Extraction ◽

Nasopharyngeal Swab ◽

Acid Extraction ◽

Rt Pcr ◽

Nucleic Acid Extraction ◽

Suitable Alternative ◽

Pcr Inhibitors ◽

Pcr Targeting

SARS-CoV-2 RT-PCR with pooled specimens has been implemented during the COVID-19 pandemic as a cost- and manpower-saving strategy for large-scale testing. However, there is a paucity of data on the efficiency of different nucleic acid extraction platforms on pooled specimens. This study compared a novel automated high-throughput liquid-based RNA extraction (LRE) platform (PHASIFYTM) with a widely used magnetic bead-based total nucleic acid extraction (MBTE) platform (NucliSENS® easyMAG®). A total of 60 pools of nasopharyngeal swab and 60 pools of posterior oropharyngeal saliva specimens, each consisting of 1 SARS-CoV-2 positive and 9 SARS-CoV-2 negative specimens, were included for the comparison. Real-time RT-PCR targeting the SARS-CoV-2 RdRp/Hel gene was performed, and GAPDH RT-PCR was used to detect RT-PCR inhibitors. No significant differences were observed in the Ct values and overall RT-PCR positive rates between LRE and MBTE platforms (92.5% (111/120] vs 90% (108/120]), but there was a slightly higher positive rate for LRE (88.3% (53/60]) than MBTE (81.7% (49/60]) among pooled saliva. The automated LRE method is comparable to a standard MBTE method for the detection of SAR-CoV-2 in pooled specimens, providing a suitable alternative automated extraction platform. Furthermore, LRE may be better suited for pooled saliva specimens due to more efficient removal of RT-PCR inhibitors.

Download Full-text

Synergistic use of electroosmotic flow and magnetic forces for nucleic acid extraction

The Analyst ◽

10.1039/c9an02191d ◽

2020 ◽

Vol 145 (6) ◽

pp. 2412-2419 ◽

Cited By ~ 1

Author(s):

Rachel N. Deraney ◽

Lindsay Schneider ◽

Anubhav Tripathi

Keyword(s):

Nucleic Acid ◽

Electric Field ◽

Microfluidic Chip ◽

Applied Electric Field ◽

Electroosmotic Flow ◽

Acid Extraction ◽

Nucleic Acid Extraction ◽

Magnetic Forces ◽

Extraction And Purification

NA extraction and purification utilitzing a microfluidic chip with applied electric field to induce electroosmotic flow opposite the magnetic NA-bound bead mix.

Download Full-text