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Comparison and optimization of DNA Isolation protocols for high throughput genomic studies of Acacia pachyceras Schwartz.

ABSTRACT: We describe the optimization and validation of six DNA isolation protocols from fresh leaves of the rare tree Acacia pachyceras. The first four protocols employed three commercial kits (Sigma, Nucleospin1, Nucleospin 2, Promega) whereas the remaining two were based on the traditional sodium dodecyl sulfate (SDS) and cetyltrimethylammonium bromide CTAB methods. Each protocol provided significantly different results concerning DNA concentration (p < 0.032), yield (p < 0.000), contaminant carry over, protocol duration, cost per sample, and comprehensive cost. We demonstrated the applicability of all the tested protocols in DNA barcoding. The protocol yielded maximum amounts (92.85 µg) of DNA in a rapid turnaround time (8 h). The quantity and purity surpassed all the other tested methods. DNA extracted by the CTAB method was the best for NGS (Phred score >Q30). These protocols will be useful tools for molecular research of Acacia pachyceras and other closely related tree species.

SUBMITTER: Habibi N

PROVIDER: S-EPMC9386096 | biostudies-literature | 2022

REPOSITORIES: biostudies-literature

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Comparison and optimization of DNA Isolation protocols for high throughput genomic studies of Acacia pachyceras Schwartz.

Habibi Nazima N Al Salameen Fadila F Rahman Muhammed M Shajan Anisha A Zakir Farhana F Abdulrazzack Nasreem N

MethodsX 20220728

We describe the optimization and validation of six DNA isolation protocols from fresh leaves of the rare tree Acacia pachyceras. The first four protocols employed three commercial kits (Sigma, Nucleospin1, Nucleospin 2, Promega) whereas the remaining two were based on the traditional sodium dodecyl sulfate (SDS) and cetyltrimethylammonium bromide CTAB methods. Each protocol provided significantly different results concerning DNA concentration (p < 0.032), yield (p < 0.000), ...[more]

PMID: 35990814

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Project description:IntroductionWhen analyzing the human plasma metabolome with Nuclear Magnetic Resonance (NMR) spectroscopy, the Carr-Purcell-Meiboom-Gill (CPMG) experiment is commonly employed for large studies. However, this process can lead to compromised statistical analyses due to residual macromolecule signals. In addition, the utilization of Trimethylsilylpropanoic acid (TSP) as an internal standard often leads to quantification issues, and binning, as a spectral summarization step, can result in features not clearly assignable to metabolites.ObjectivesOur aim was to establish a new complete protocol for large plasma cohorts collected with the purpose of describing the comparative metabolic profile of groups of samples.MethodsWe compared the conventional CPMG approach to a novel procedure that involves diffusion NMR, using the Longitudinal Eddy-Current Delay (LED) experiment, maleic acid (MA) as the quantification reference and peak picking for spectral reduction. This comparison was carried out using the ultrafiltration method as a gold standard in a simple sample classification experiment, with Partial Least Squares-Discriminant Analysis (PLS-DA) and the resulting metabolic signatures for multivariate data analysis. In addition, the quantification capabilities of the method were evaluated.ResultsWe found that the LED method applied was able to detect more metabolites than CPMG and suppress macromolecule signals more efficiently. The complete protocol was able to yield PLS-DA models with enhanced classification accuracy as well as a more reliable set of important features than the conventional CPMG approach. Assessment of the quantitative capabilities of the method resulted in good linearity, recovery and agreement with an established amino acid assay for the majority of the metabolites tested. Regarding repeatability, ~ 85% of all peaks had an adequately low coefficient of variation (< 30%) in replicate samples.ConclusionOverall, our comparison yielded a high-throughput untargeted plasma NMR protocol for optimized data acquisition and processing that is expected to be a valuable contribution in the field of metabolic biomarker discovery.

Project description:The successful translation of mesenchymal stem cells (MSCs) from bench to bedside is predicated upon their regenerative capabilities and immunomodulatory potential. Many challenges still exist in making MSCs a viable and cost-effective therapeutic option, due in part to the challenges of sourcing MSCs from adult tissues and inconsistencies in the characterization of MSCs. In many cases, adult MSC collection is an invasive procedure, and ethical concerns and age-related heterogeneity further complicate obtaining adult tissue derived MSCs at the scales needed for clinical applications. Alternative adult sources, such as post-partum associated tissues, offer distinct advantages to overcome these challenges. However, successful therapeutic applications rely on the efficient ex-vivo expansion of the stem cells while avoiding any culture-related phenotypic alterations, which requires optimized and standardized isolation, culture, and cell preservation methods. In this review, we have compared the isolation and culture methods for MSCs originating from the human amniotic membrane (hAMSCs) of the placenta to identify the elements that support the extended subculture potential of hAMSCs without compromising their immune-privileged, pluripotent regenerative potential.Abbreviations: AM: Human amniotic membrane; ASCs: Adipose tissue-derived stem cells; BM-MSCs: Bone marrow-mesenchymal stem cells; DMEM: Dulbecco's modified eagle medium; DT: Doubling time; EMEM: Eagle's modified essential medium; ESCM: Embryonic stem cell markers; ESCs: Embryonic stem cells; hAECs: Human amniotic epithelial cells; hAMSCs: Human amniotic mesenchymal stem cells; HLA: Human leukocyte antigen; HM: Hematopoietic markers; IM: Immunogenicity markers; MHC: Major histocompatibility complex; MSCs: Mesenchymal stem cells; MCSM: Mesenchymal cell surface markers; Nanog: NANOG homeobox; Oct: Octamer binding transcription factor 4; P: Passage; PM: Pluripotency markers; STRO-1: Stromal precursor antigen-1; SCP: Subculture potential; Sox-2: Sry-related HMG box gene 2; SSEA-4: Stage-specific embryonic antigen; TRA: Tumor rejection antigen.

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Comparison and optimization of DNA Isolation protocols for high throughput genomic studies of Acacia pachyceras Schwartz.

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Comparison and optimization of DNA Isolation protocols for high throughput genomic studies of <i>Acacia pachyceras</i> Schwartz.

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