The Integrity and Yield of Genomic DNA Isolated from Whole Blood Following Long-Term Storage at −30°C

Background: Though blood is an excellent biofluid for metabolomics, proteins and lipids present in blood can interfere with 1d-1H NMR spectra and disrupt quantification of metabolites. Here, we present effective macromolecule removal strategies for serum and whole blood (WB) samples. Methods: A variety of macromolecule removal strategies were compared in both WB and serum, along with tests of ultrafiltration alone and in combination with precipitation methods. Results: In healthy human serum, methanol:chloroform:water extraction with ultrafiltration was compared to methanol precipitation with and without ultrafiltration. Methods were tested in healthy pooled human serum, and in serum from patients with sepsis. Effects of long-term storage at −80 °C were tested to explore the impact of macromolecule removal strategy on serum from different conditions. In WB a variety of extraction strategies were tested in two types of WB (from pigs and baboons) to examine the impact of macromolecule removal strategies on different samples. Conclusions: In healthy human serum methanol precipitation of serum with ultrafiltration was superior, but was similar in recovery and variance to methanol:chloroform:water extraction with ultrafiltration in pooled serum from patients with sepsis. In WB, high quality, quantifiable spectra were obtained with the use of a methanol: chloroform precipitation.

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Capture and Purification of Plant Genomic DNA on a Readily-available Cellulose Matrix

10.1101/163212 ◽

2017 ◽

Author(s):

Megan M. Thompson ◽

Estelle M. Hrabak

Keyword(s):

Arabidopsis Thaliana ◽

Nucleic Acids ◽

Plant Species ◽

Efficient Method ◽

Genomic Dna ◽

Low Cost ◽

Long Term Storage ◽

Cellulose Matrix ◽

Term Storage

AbstractWhatman FTA ®Cards are a fast and efficient method for capturing and storing nucleic acids but are cost-prohibitive for some researchers. We developed a method that substitutes readily-available cellulose-based paper and homemade washing buffer for commercial FTA ®Cards and FTA ®Purification Reagent. This method is suitable for long-term storage of DNA from many plant species, including Arabidopsis thaliana, prior to purification and PCR.Method SummaryHere we report a low-cost method for long-term storage of plant genomic DNA on a readily available cellulose matrix.

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Integrating Whole Blood Transcriptomic Collection Procedures Into the Current Anti-Doping Testing System, Including Long-Term Storage and Re-Testing of Anti-Doping Samples

Frontiers in Molecular Biosciences ◽

10.3389/fmolb.2021.728273 ◽

2021 ◽

Vol 8 ◽

Author(s):

Giscard Lima ◽

Alexander Kolliari-Turner ◽

Fernanda Rossell Malinsky ◽

Fergus M. Guppy ◽

Renan Paulo Martin ◽

...

Keyword(s):

Whole Blood ◽

Differentially Expressed ◽

Rna Seq ◽

Short Term ◽

Long Term Storage ◽

Short Term Storage ◽

Short And Long Term ◽

Rna Preservation ◽

Term Storage

Introduction: Recombinant human erythropoietin (rHuEPO) administration studies involving transcriptomic approaches have demonstrated a gene expression signature that could aid blood doping detection. However, current anti-doping testing does not involve collecting whole blood into tubes with RNA preservative. This study investigated if whole blood in long-term storage and whole blood left over from standard hematological testing in short-term storage could be used for transcriptomic analysis despite lacking RNA preservation.Methods: Whole blood samples were collected from twelve and fourteen healthy nonathletic males, for long-term and short-term storage experiments. Long-term storage involved whole blood collected into Tempus™ tubes and K2EDTA tubes and subjected to long-term (i.e., ‒80°C) storage and RNA extracted. Short-term storage involved whole blood collected into K2EDTA tubes and stored at 4°C for 6‒48 h and then incubated at room temperature for 1 and 2 h prior to addition of RNA preservative. RNA quantity, purity, and integrity were analyzed in addition to RNA-Seq using the MGI DNBSEQ-G400 on RNA from both the short- and long-term storage studies. Genes presenting a fold change (FC) of >1.1 or < ‒1.1 with p ≤ 0.05 for each comparison were considered differentially expressed. Microarray analysis using the Affymetrix GeneChip® Human Transcriptome 2.0 Array was additionally conducted on RNA from the short-term study with a false discovery ratio (FDR) of ≤0.05 and an FC of >1.1 or < ‒1.1 applied to identify differentially expressed genes.Results: RNA quantity, purity, and integrity from whole blood subjected to short- and long-term storage were sufficient for gene expression analysis. Long-term storage: when comparing blood tubes with and without RNA preservation 4,058 transcripts (6% of coding and non-coding transcripts) were differentially expressed using microarray and 658 genes (3.4% of mapped genes) were differentially expressed using RNA-Seq. Short-term storage: mean RNA integrity and yield were not significantly different at any of the time points. RNA-Seq analysis revealed a very small number of differentially expressed genes (70 or 1.37% of mapped genes) when comparing samples stored between 6 and 48 h without RNA preservative. None of the genes previously identified in rHuEPO administration studies were differently expressed in either long- or short-term storage experiments.Conclusion: RNA quantity, purity, and integrity were not significantly compromised from short- or long-term storage in blood storage tubes lacking RNA stabilization, indicating that transcriptomic analysis could be conducted using anti-doping samples collected or biobanked without RNA preservation.

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Effects of long‐term storage and delayed hemolysis of whole blood on folate species by LC‐MS/MS

The FASEB Journal ◽

10.1096/fasebj.23.1_supplement.557.1 ◽

2009 ◽

Vol 23 (S1) ◽

Author(s):

Zia Fazili‐Qari ◽

Christine M Pfeiffer ◽

Mindy Zhang

Keyword(s):

Whole Blood ◽

Long Term Storage ◽

Term Storage

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Studies on genetic changes in rye samples (Secale cereale L.) maintained in a seed bank

Cellular & Molecular Biology Letters ◽

10.2478/s11658-006-0028-0 ◽

2006 ◽

Vol 11 (3) ◽

Cited By ~ 8

Author(s):

Katarzyna Chwedorzewska ◽

Piotr Bednarek ◽

Renata Lewandowska ◽

Paweł Krajewski ◽

Jerzy Puchalski

Keyword(s):

Genomic Dna ◽

Fragment Length Polymorphism ◽

Gene Bank ◽

Genetic Changes ◽

Long Term Storage ◽

Seed Ageing ◽

Secale Cereale L ◽

Principle Coordinate Analysis ◽

Term Storage

AbstractThe aim of this study was to identify genetic changes in rye seeds induced by natural ageing during long-term storage and consecutive regeneration cycles under gene bank conditions. Genomic DNA from four rye samples varying in their initial viability after one and three cycles of reproduction was analyzed by AFLP (amplified fragment length polymorphism) fingerprinting. Seven EcoRI/MseI primer combinations defined 663 fragments, and seven PstI/MseI primer combinations defined 551 fragments. The variation in the frequency of the seventy-four EcoRI/MseI bands was statistically significant between samples. These changes could be attributed to genetic changes occurring during storage and regeneration. However, the PstI/MseI fragments appeared to be uninfluenced by seed ageing, regeneration and propagation. A combined Principle Coordinate Analysis revealed differences between samples with different initial viability. We showed that materials with low initial viability differ in their response from highly viable ones, and that the changes exhibited in the former case are preserved through regeneration cycles.

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A feasible method to extract DNA from the cambium of high-canopy trees: from harvest to assessment

Acta Amazonica ◽

10.1590/1809-4392202001571 ◽

2020 ◽

Vol 50 (4) ◽

pp. 335-338

Author(s):

Érica MANGARAVITE ◽

Vanessa TERRA ◽

Eric Koiti Okiyama HATTORI ◽

Thaís Carolina da Silva DAL’SASSO ◽

Leonardo Lopes BHERING ◽

...

Keyword(s):

Genomic Dna ◽

Storage Conditions ◽

Microsatellite Genotyping ◽

Short Term ◽

Feasible Method ◽

Long Term Storage ◽

Canopy Trees ◽

Amazonian Forests ◽

Term Storage

ABSTRACT Many tropical trees have high canopies and their leaves are not accessible. Thus, the use of tissue from a more accessible organ (cambium) for DNA extraction may be an alternative for molecular studies. We adapted a feasible methodology for extracting genomic DNA from cambium tissue harvested in the field for the assessment with PCR. We tested three storage conditions (two buffers and a silica gel) and four periods of time after harvest. We used previously described protocols and tested them on three species that occur in Amazonian forests and other biomes: Anadenanthera peregrina var. peregrina, Cedrela fissilis, and Ceiba speciosa. Our protocol obtained suitable PCR-grade genomic DNA for DNA sequencing and microsatellite genotyping. We recommend the use of silica for long-term storage and the buffer with ascorbic acid for short-term storage.

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Long-term storage of blood samples as whole blood at extremely low temperatures for methemoglobin determination

Forensic Science International ◽

10.1016/0379-0738(88)90098-9 ◽

1988 ◽

Vol 37 (2) ◽

pp. 99-104 ◽

Cited By ~ 10

Author(s):

Keizo Sato ◽

Keiji Tamaki ◽

Hiroshi Okajima ◽

Yoshinao Katsumata

Keyword(s):

Whole Blood ◽

Low Temperatures ◽

Blood Samples ◽

Long Term Storage ◽

Term Storage

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Long-term storage of blood prior to whole-blood lymphocyte culturing

Veterinary Immunology and Immunopathology ◽

10.1016/0165-2427(81)90015-5 ◽

1981 ◽

Vol 2 (6) ◽

pp. 555-560 ◽

Cited By ~ 1

Author(s):

J.M.B. Kaneene ◽

F. Soper ◽

D.W. Johnson ◽

R.K. Anderson

Keyword(s):

Whole Blood ◽

Blood Lymphocyte ◽

Long Term Storage ◽

Term Storage

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Shelf life of whole blood samples in a biobank and the yield of deoxyribonucleic acid during genetic testing

CARDIOVASCULAR THERAPY AND PREVENTION ◽

10.15829/1728-8800-2020-2726 ◽

2020 ◽

Vol 19 (6) ◽

pp. 2726

Author(s):

O. P. Skirko ◽

A. N. Meshkov ◽

I. A. Efimova ◽

V. A. Kutsenko ◽

A. V. Kiseleva ◽

...

Keyword(s):

Shelf Life ◽

Whole Blood ◽

Deoxyribonucleic Acid ◽

Blood Samples ◽

Dna Concentration ◽

National Medical ◽

Long Term Storage ◽

Whole Blood Samples ◽

Term Storage

Aim. To study the effect of the shelf life of frozen whole blood samples in a biobank on the amount of released deoxyribonucleic acid (DNA).Material and methods. The study included whole blood samples placed in tubes with the anticoagulant EDTA (ethylenediaminetetraacetic acid at a concentration of 1,8 mg/ml) from participants in the epidemiological study ESSE-RF-1 and ESSE-RF-2 and cohort studies conducted at the National Medical Research Center for Therapy and Preventive Medicine. The samples were stored in the biobank of the National Medical Research Center for Therapy and Preventive Medicine at temperature from -22О C to -32О C. The shelf life from blood collection to DNA extraction ranged from several weeks to 11 years. DNA was extracted using QIAamp DNA Blood Mini Kit (250) and 96 Blood Kit (Qiagen, Germany). Statistical analysis was performed using the R 3.6.1 software. To analyze the association of blood storage time with the logarithm of DNA concentration, a linear regression was used.Results. The analysis included data on the DNA concentration of 5405 samples. Multivariate regression showed that the blood shelf life was significantly associated with a decrease in concentration by 3,92% (3,16-4,68) for each year of storage (p <0,0001). For 509 samples, the DNA concentration was measured twice, immediately after isolation and after 4,5 years of DNA storage at -32О C. During storage, the concentration of DNA increased by an average of 2% (p=0,046).Conclusion. Long-term storage of whole blood samples at temperature from -22О C to -32О C is associated with a decrease in the DNA yield. Long-term storage of the isolated DNA at a temperature of -32О C is not associated with a decrease in its concentration.

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Comparison of ex vivo stability of copeptin and vasopressin

Clinical Chemistry and Laboratory Medicine (CCLM) ◽

10.1515/cclm-2016-0559 ◽

2017 ◽

Vol 55 (7) ◽

Cited By ~ 5

Author(s):

Judith E. Heida ◽

Lianne S.M. Boesten ◽

Esmée M. Ettema ◽

Anneke C. Muller Kobold ◽

Casper F.M. Franssen ◽

...

Keyword(s):

Whole Blood ◽

Storage Temperature ◽

Ex Vivo ◽

Freezing And Thawing ◽

Short Term ◽

Long Term Storage ◽

Repeated Freezing ◽

Vasopressin Precursor ◽

Term Storage

AbstractBackground:Copeptin, part of the vasopressin precursor, is increasingly used as marker for vasopressin and is claimed to have better ex vivo stability. However, no study has directly compared the ex vivo stability of copeptin and vasopressin.Methods:Blood of ten healthy volunteers was collected in EDTA tubes. Next, we studied the effect of various pre-analytical conditions on measured vasopressin and copeptin levels: centrifugation speed, short-term storage temperature and differences between whole blood and plasma, long-term storage temperature and repeated freezing and thawing. The acceptable change limit (ACL), indicating the maximal percentage change that can be explained by assay variability, was used as cut-off to determine changes in vasopressin and copeptin.Results:The ACL was 25% for vasopressin and 19% for copeptin. Higher centrifugation speed resulted in lower vasopressin levels, whereas copeptin concentration was unaffected. In whole blood, vasopressin was stable up to 2 h at 25°C and 6 h at 4°C. In plasma, vasopressin was stable up to 6 h at 25°C and 24 h at 4°C. In contrast, copeptin was stable in whole blood and plasma for at least 24h at both temperatures. At –20°C, vasopressin was stable up to 1 month and copeptin for at least 4 months. Both vasopressin and copeptin were stable after 4 months when stored at –80°C and –150°C. Vasopressin concentration decreased after four freeze-thaw cycles, whereas copeptin concentration was unaffected.Conclusion:Vasopressin levels were considerably affected by pre-analytical conditions, while copeptin levels were stable. Therefore, a strict sample handling protocol for measurement of vasopressin is recommended.

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