Comparison of DNA Extraction Methods for Samples from Old Blood Collections

Tagliaferro, Sarah Samut; Zejnelagic, Azra; Farrugia, Rosienne; Wettinger, Stephanie Bezzina

doi:10.2144/btn-2020-0113

Cited by 10 publications

(6 citation statements)

References 13 publications

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“…Firstly, whole blood samples were collected from patients and kept frozen at -20°C for six months. While fresh samples are producing better results, it is not unusual to produce high-quality DNA isolated from samples that have been stored for several years (Tagliaferro et al, 2021). Also, QIAamp® DNA Mini Kit that was used in the present study is best valued for its time-effectiveness, lower possibility of sample contamination and ease of use (Chacon-Cortes & Griffiths, 2014).…”

Section: Resultsmentioning

confidence: 99%

“…However, previous research has shown that for older samples, other DNA extraction methods, such as modified salting-out method, should be used (Mardan-Nik et al, 2019). Other studies have shown that silica-based DNA isolation using commercial kits, such as QIAamp® DNA Blood Midi Kit (Qiagen) (Mardan-Nik et al, 2019) and DNeasy® Blood & Tissue Kit (Qiagen) (Tagliaferro et al, 2021) are both capable of producing high-quality DNA extracts suitable for subsequent PCR amplification.…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Challenges in obtaining high-quality data from a custom-made panel for the next generation sequencing (NGS) using Ion Torrent GeneStudio™ S5 platform

Salihefendić¹,

Ašić²,

Čeko

et al. 2022

J. bioanthropol. (Online)

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The goal of this part of the study was to optimize the sequencing procedure for 16 human genes and their regulatory regions that might be associated with differential immunological response to COVID-19. The study was performed on 60 COVID-19 patients from the General Hospital of Tešanj, Bosnia and Herzegovina, categorized into three groups of mild, moderate, and severe clinical manifestation, based on the diagnosis by the residential physician. Target coding sequences and their regulatory regions were amplified for the following genes: HLA-A, HLA-B, HLA-C, ACE2, IL-6, IL-4, TMPRSS2, IFITM3, IL-12, RIG-I/DDX58, IRF-7, IRF-9, IL-1B, IL-1A, CD55, and TNF-α. DNA was isolated from the whole blood samples stored at -20°C for six months using QIAamp® DNA Mini Kit according to manufacturer’s instructions. Since NGS analysis of target genomic regions was performed on the Ion Torrent GeneStudio™ S5 platforms, libraries were prepared using Ion AmpliSeq™ Library Kit Plus according to manufacturer’s instructions in a protocol optimized for low-quality DNA. Due to dissatisfactory sequencing results, further protocol optimization steps were employed through separating two primer pools, increasing the number of PCR cycles, and decreasing the annealing temperature for the primer pool which showed poorer amplification results. In the end, 36 samples produced optimal results, while the remaining 24 samples will be re-sequenced following repeated sample collection and DNA isolation, accompanied by additional protocol modifications.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Challenges in obtaining high-quality data from a custom-made panel for the next generation sequencing (NGS) using Ion Torrent GeneStudio™ S5 platform

Salihefendić¹,

Ašić²,

Čeko

et al. 2022

J. bioanthropol. (Online)

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show abstract

“…It is worth noting that frozen BCCs were used in most of the studies, while commercial DNA extraction kits are routinely used for fresh samples. 27 Frozen BCCs were thawed at 4°C, room temperature, 37°C, 55°C, and 65°C before DNA extraction in previous studies. 1,7,8,10,28,29 We explored three thawing conditions.…”

Section: Discussionmentioning

confidence: 99%

Evaluation of DNA extracted from residual blood clots after serological testing

Zhang,

Cheng,

Yang

et al. 2024

Cell Biochemistry & Function

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DNA quality is of paramount importance for molecular biology research. This study aimed to assess the DNA extracted from residual blood clots after serological testing, focusing on the impact of blood clot segments, extraction kits, temporary storage durations (TSDs), and thawing methods on DNA quality. We divided the residual blood clot column (BCC) from healthy donors into three segments and utilized two different extraction kits. The BCCs were subjected to four TSDs at 4°C (7 days, 10 days, 1 month, and 2 months) and three thawing methods (4°C, room temperature, and 37°C). We found that the TIANamp Blood Clot DNA Kit yielded consistently high‐quality DNA from each segment with stable A260/280 and A260/230 ratios. The DNA yield showed a strong positive correlation with leukocyte concentration, and a satisfactory median DNA yield of 28.79 μg/g BCC was obtained across all segments. DNA integrity, as measured by the DNA integrity number and DNA fragment peak size, decreased with increasing TSD at 4°C, with a notable decrease after 10 days of storage. Thawing at 37°C resulted in the lowest DNA fragment peak size. In conclusion, BCC could be an ideal DNA source with satisfactory yield and purity. A prolonged TSD at 4°C leads to an obvious decrease in DNA integrity, and thawing the frozen BCC at 37°C decreases DNA fragment sizes. To maintain DNA integrity, BCCs should be cryopreserved as soon as possible after short TSDs at 4°C and thawed at 4°C.

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“…Currently, EV‐DNA isolation methods are not standardized, and are based on either commercially available kits (i.e. silica filtration and magnetic beads‐based approach) or phenol chloroform method which can affect isolation efficiency and DNA fragment size (Sorber et al., 2017; Tagliaferro et al., 2021). Downstream analysis methods were also reported, such as Qubit fluorometer, nanodrop, Agilent bioanalyzer and qPCR, which present different levels of sensitivity and specificity.…”

Section: Discussionmentioning

confidence: 99%

EV‐ADD, a database for EV‐associated DNA in human liquid biopsy samples

Tsering

Chen

et al. 2022

J of Extracellular Vesicle

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Extracellular vesicles (EVs) play a key role in cellular communication both in physiological conditions and in pathologies such as cancer. Emerging evidence has shown that EVs are active carriers of molecular cargo (e.g. protein and nucleic acids) and a powerful source of biomarkers and targets. While recent studies on EV‐associated DNA (EV‐DNA) in human biofluids have generated a large amount of data, there is currently no database that catalogues information on EV‐DNA. To fill this gap, we have manually curated a database of EV‐DNA data derived from human biofluids (liquid biopsy) and in‐vitro studies, called the Extracellular Vesicle‐Associated DNA Database (EV‐ADD). This database contains validated experimental details and data extracted from peer‐reviewed published literature. It can be easily queried to search for EV isolation methods and characterization, EV‐DNA isolation techniques, quality validation, DNA fragment size, volume of starting material, gene names and disease context. Currently, our database contains samples representing 23 diseases, with 13 different types of EV isolation techniques applied on eight different human biofluids (e.g. blood, saliva). In addition, EV‐ADD encompasses EV‐DNA data both representing the whole genome and specifically including oncogenes, such as KRAS, EGFR, BRAF, MYC, and mitochondrial DNA (mtDNA). An EV‐ADD data metric system was also integrated to assign a compliancy score to the MISEV guidelines based on experimental parameters reported in each study. While currently available databases document the presence of proteins, lipids, RNA and metabolites in EVs (e.g. Vesiclepedia, ExoCarta, ExoBCD, EVpedia, and EV‐TRACK), to the best of our knowledge, EV‐ADD is the first of its kind to compile all available EV‐DNA datasets derived from human biofluid samples. We believe that this database provides an important reference resource on EV‐DNA‐based liquid biopsy research, serving as a learning tool and to showcase the latest developments in the EV‐DNA field. EV‐ADD will be updated yearly as newly published EV‐DNA data becomes available and it is freely available at http://www.evdnadatabase.com.

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Comparison of DNA Extraction Methods for Samples from Old Blood Collections

Cited by 10 publications

References 13 publications

Challenges in obtaining high-quality data from a custom-made panel for the next generation sequencing (NGS) using Ion Torrent GeneStudio™ S5 platform

Challenges in obtaining high-quality data from a custom-made panel for the next generation sequencing (NGS) using Ion Torrent GeneStudio™ S5 platform

Evaluation of DNA extracted from residual blood clots after serological testing

EV‐ADD, a database for EV‐associated DNA in human liquid biopsy samples

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