scholarly journals Optimized protocol for the extraction of RNA and DNA from frozen whole blood sample stored in a single EDTA tube

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Hirotaka Yamagata ◽  
Ayumi Kobayashi ◽  
Ryouichi Tsunedomi ◽  
Tomoe Seki ◽  
Masaaki Kobayashi ◽  
...  
Keyword(s):  
Whole Blood ◽  
Ethylenediaminetetraacetic Acid ◽  
Rna Extraction ◽  
Basic Research ◽  
Rna Degradation ◽  
Disodium Salt ◽  
Water Bath ◽  
High Quality ◽  
Blood Samples ◽  
Rna And Dna

AbstractCryopreservation of whole blood is useful for DNA collection, and clinical and basic research. Blood samples in ethylenediaminetetraacetic acid disodium salt (EDTA) tubes stored at − 80 °C are suitable for DNA extraction, but not for high-quality RNA extraction. Herein, a new methodology for high-quality RNA extraction from human blood samples is described. Quickly thawing frozen whole blood on aluminum blocks at room temperature could minimize RNA degradation, and improve RNA yield and quality compared with thawing the samples in a 37 °C water bath. Furthermore, the use of the NucleoSpin RNA kit increased RNA yield by fivefold compared with the PAXgene Blood RNA Kit. Thawing blood samples on aluminum blocks significantly increased the DNA yield by ~ 20% compared with thawing in a 37 °C water bath or on ice. Moreover, by thawing on aluminum blocks and using the NucleoSpin RNA and QIAamp DNA Blood kits, the extraction of RNA and DNA of sufficient quality and quantity was achieved from frozen EDTA whole blood samples that were stored for up to 8.5 years. Thus, extracting RNA from frozen whole blood in EDTA tubes after long-term storage is feasible. These findings may help advance gene expression analysis, as well as biomarker research for various diseases.

scholarly journals Detection of Babesia RNA and DNA in whole blood samples from US blood donations

Transfusion ◽  
2021 ◽  
Author(s):  
Jean Stanley ◽  
Susan L. Stramer ◽  
Yasuko Erickson ◽  
Julie Cruz ◽  
Jed Gorlin ◽  
...  
Keyword(s):  
Whole Blood ◽  
Blood Donations ◽  
Blood Samples ◽  
Whole Blood Samples ◽  
Rna And Dna

scholarly journals A comprehensive RNA handling and transcriptomics guide for high-throughput processing of Plasmodium blood-stage samples

2020 ◽  
Vol 19 (1) ◽  
Author(s):  
Michal Kucharski ◽  
Jaishree Tripathi ◽  
Sourav Nayak ◽  
Lei Zhu ◽  
Grennady Wirjanata ◽  
...  
Keyword(s):  
High Throughput ◽  
Large Scale ◽  
Rna Extraction ◽  
Transcriptome Data ◽  
High Quality ◽  
Diverse Range ◽  
Human Haemoglobin ◽  
Infected Blood ◽  
Blood Samples ◽  
Total Rna

Abstract Background Sequencing technology advancements opened new opportunities to use transcriptomics for studying malaria pathology and epidemiology. Even though in recent years the study of whole parasite transcriptome proved to be essential in understanding parasite biology there is no compiled up-to-date reference protocol for the efficient generation of transcriptome data from growing number of samples. Here, a comprehensive methodology on how to preserve, extract, amplify, and sequence full-length mRNA transcripts from Plasmodium-infected blood samples is presented that can be fully streamlined for high-throughput studies. Results The utility of various commercially available RNA-preserving reagents in a range of storage conditions was evaluated. Similarly, several RNA extraction protocols were compared and the one most suitable method for the extraction of high-quality total RNA from low-parasitaemia and low-volume blood samples was established. Furthermore, the criteria needed to evaluate the quality and integrity of Plasmodium RNA in the presence of human RNA was updated. Optimization of SMART-seq2 amplification method to better suit AT-rich Plasmodium falciparum RNA samples allowed us to generate high-quality transcriptomes from as little as 10 ng of total RNA and a lower parasitaemia limit of 0.05%. Finally, a modified method for depletion of unwanted human haemoglobin transcripts using in vitro CRISPR-Cas9 treatment was designed, thus improving parasite transcriptome coverage in low parasitaemia samples. To prove the functionality of the pipeline for both laboratory and field strains, the highest  2-hour resolution RNA-seq transcriptome for P. falciparum 3D7 intraerythrocytic life cycle available to  date was generated, and the entire protocol was applied to create the largest transcriptome data from Southeast Asian field isolates. Conclusions Overall, the presented methodology is an inclusive pipeline for generation of good quality transcriptomic data from a diverse range of Plasmodium-infected blood samples with varying parasitaemia and RNA inputs. The flexibility of this pipeline to be adapted to robotic handling will facilitate both small and large-scale future transcriptomic studies in the field of malaria.

Rapid Purification of High Quality DNA from Large Whole Blood Samples Using a New Rapid Protocol

2004 ◽  
Vol 4 (4) ◽  
pp. 372-374 ◽  
Author(s):  
J.-R. Long ◽  
Y.-Y. Zhang ◽  
E. Hofer ◽  
C. Seemann ◽  
M. Schatz ◽  
...  
Keyword(s):  
Whole Blood ◽  
High Quality ◽  
Blood Samples ◽  
Whole Blood Samples ◽  
Rapid Purification

scholarly journals Effect of RNA preservation methods on RNA quantity and quality of field collected avian whole blood

2021 ◽  
Author(s):  
Johanna A Harvey ◽  
Sarah A Knutie
Keyword(s):  
Active Material ◽  
Rna Extraction ◽  
Quality Measures ◽  
Rna Degradation ◽  
Blood Samples ◽  
Dry Ice ◽  
Total Rna ◽  
Rna Integrity ◽  
Avian Blood ◽  
Rna Preservation

A limitation of comparative transcriptomic studies of wild avian populations continues to be sample acquisition and preservation to achieve resulting high-quality RNA (i.e., ribonucleic acids that transfers, translates, and regulates the genetic code from DNA into proteins). Field sampling of wild bird samples provides challenges as RNA degradation progresses quickly and because cryopreservation is often not feasible at remote locations. We collected blood samples from songbirds, as avian blood is nucleated and provides sufficient transcriptionally active material in a small and non-lethal sample, to compare the efficacy of widely available RNA stabilizing buffers, RNAlater (Ambion) and DNA/RNA Shield (Zymo) at differing concentrations along with a dry ice-based flash freezing method (Isopropanol 99% and dry ice mixture, -109°C). Each blood sample was divided among five different preservation treatments (dry ice-based flash freezing, RNAlater with 1:5 or 1:10 dilution, or DNA/RNA Shield with 1:2 or 1:3 dilution). A new protocol was optimized for total RNA extraction from avian blood samples with small starting volumes enabling sampling of small passerines. We quantified quality measures, RNA integrity numbers (RINe), rRNA ratios, and total RNA concentrations. We found that RNA preservation buffers, RNAlater and DNA/RNA Shield at all concentrations, provide sample protection from RNA degradation. We suggest caution against using dry ice-based flash-freezing alone for samples preservation as these samples resulted in lower quality measures then samples in preservation buffer. Total RNA concentration was generally not affected by preservation treatment and may vary due to differences in initial samples volumes and carryover across processing steps.

scholarly journals Stabilization of gene expression in whole blood: high quality RNA with room temperature handling of human blood samples.

2013 ◽  
Vol 27 (S1) ◽  
Author(s):  
Vasco Liberal ◽  
Angela Stassinopoulos ◽  
Scott Whitney ◽  
Steven Wilkinson ◽  
Winnie Huang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Human Blood ◽  
Whole Blood ◽  
Room Temperature ◽  
High Quality ◽  
Blood Samples ◽  
Human Blood Samples

scholarly journals Factors Influencing Degradation Kinetics of mRNAs And Half-Lives of MicroRNAs, circRNAs, lncRNAs In Blood In Vitro Using Quantitative PCR

2021 ◽  
Author(s):  
Chong Wang ◽  
Hui Liu
Keyword(s):  
Quantitative Pcr ◽  
Room Temperature ◽  
Degradation Kinetics ◽  
Rna Extraction ◽  
Rna Degradation ◽  
Circular Rnas ◽  
Orthogonal Experimental Design ◽  
Messenger Rnas ◽  
Storage Duration ◽  
Blood Samples

Abstract Background: RNAs are rapidly degraded in samples and during collection, processing and testing. In this study, we used the same method to explore the half-lives of different RNAs and the influencing factors, and compared the degradation kinetics and characteristics of different RNAs in whole blood and experimental samples.Methods: Fresh anticoagulant blood samples were incubated at room temperature for different durations, RNAs were extracted, and genes, including internal references, were amplified by real-time quantitative PCR. A linear half-life model was established according to cycle threshold (Ct) values. The effects of experimental operations on RNA degradation before and after RNA extraction were explored. Quantitative analysis of mRNA degradation in samples and during experimental processes were explored using an orthogonal experimental design.Results: The storage duration of blood samples at room temperature had the greatest influence on RNA degradation. The half-lives of messenger RNAs (mRNAs) was 16.4 h. The half-lives of circular RNAs (circRNAs), long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) were 27.6h, 21.1h and 11.4h, respectively.Conclusion: RNA degradation occurred mainly in blood samples, and the half-lives of mRNAs and miRNAs were similar. Quantitative experiments related to mRNAs should be completed within 2 h. The half-lives of circRNAs and lncRNAs were longer than those of the former two.

scholarly journals Effects of preservation duration at 4 °C on the quality of RNA in rabbit blood specimens

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e8940
Author(s):  
Jiaojiao Song ◽  
Junmei Zhou
Keyword(s):  
Whole Blood ◽  
Rna Degradation ◽  
High Quality ◽  
Multicenter Studies ◽  
Rna Quality ◽  
Rabbit Blood ◽  
Significant Change ◽  
Blood Specimens ◽  
Storage Facilities

A prolonged preservation duration of blood specimens at 4 °C may occur due to the distance from collection points to storage facilities in many biobanks, especially for multicenter studies. This could lead to RNA degradation, affecting downstream analyses. However, effects of preservation durations at 4 °C on RNA quality in blood specimens need to be studied. We collected rabbit blood using EDTA tubes and stored them at 4 °C for different preservation durations. Then, we examined the quality of RNA from whole blood and leukocytes isolated from rabbit blood. Our results show that the purity of whole blood RNA and leukocyte RNA does not indicate significant change after rabbit blood is stored at 4 °C for different preservation durations (from 1 h to 7 days). The integrity of leukocyte RNA indicates the same result as above, but the integrity of whole blood RNA is significantly decreased after rabbit blood is stored at 4 °C for over 3 days. Moreover, expression of SMAD7, MKI67, FOS, TGFβ1 and HIF1α of whole blood RNA and leukocyte RNA remains basically stable, but PCNA expression of whole blood RNA or leukocyte RNA is significantly decreased after rabbit blood is stored at 4 °C for over 24 h or 7 days. Therefore, these results suggest that high-quality RNA is obtained from the fresher blood specimens and if blood specimens are stored for over 3 days at 4 °C, the quality of leukocyte RNA is more stable and of better quality than that of whole blood RNA.

scholarly journals Fully Automated Detection of Hepatitis C Virus RNA in Serum and Whole-Blood Samples

2002 ◽  
Vol 9 (6) ◽  
pp. 1385-1388 ◽  
Author(s):  
Harald H. Kessler ◽  
Alexandra M. K. Clarici ◽  
Evelyn Stelzl ◽  
Gerhard Mühlbauer ◽  
Elisabeth Daghofer ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Whole Blood ◽  
Rna Extraction ◽  
Hcv Rna ◽  
Molecular Assay ◽  
Blood Samples ◽  
Molecular Assays ◽  
Roche Molecular Diagnostics ◽  
Whole Blood Samples

ABSTRACT In this study, we established a fully automated molecular assay for qualitative detection of hepatitis C virus (HCV) in serum and whole-blood samples and compared it with conventional molecular assays, including manual HCV RNA extraction protocols. Whole-blood samples were collected from patients with and without chronic HCV infection in EDTA tubes and nucleic acid stabilization tubes (NASTs). Prior to HCV RNA extraction, the HCV Internal Control (IC), derived from the COBAS AMPLICOR HCV test, version 2.0 (Roche Molecular Diagnostics), was added. The new assay was based on an automated extraction protocol on the MagNA Pure LC instrument (Roche Applied Science), followed by automated reverse transcription, amplification, hybridization, and detection on the Cobas Amplicor analyzer (Roche Molecular Diagnostics). The detection limit of the new assay was found to be similar to those of conventional molecular assays. In clinical samples, 100% agreement between the new assay and conventional methods was observed. The introduced amount of IC was detected in 45 of 45 serum samples, 41 of 45 EDTA tube whole-blood samples, and 43 of 45 NAST whole-blood samples. Retesting led to more frequent IC detection. The fully automated molecular assay was found to be suitable for detection of HCV RNA in different kinds of sample materials. It may be recommended for use in the high-throughput routine molecular diagnostic laboratory.

scholarly journals The effectiveness of cooling conditions on temperature of canine EDTA whole blood samples

PeerJ ◽  
2016 ◽  
Vol 4 ◽  
pp. e2732
Author(s):  
Karen M. Tobias ◽  
Leslie Serrano ◽  
Xiaocun Sun ◽  
Bente Flatland
Keyword(s):  
Whole Blood ◽  
Bath Temperature ◽  
Water Bath ◽  
Ammonium Concentration ◽  
Blood Samples ◽  
Treatment Conditions ◽  
Laboratory Test Results ◽  
Rate Of Cooling ◽  
Whole Blood Samples ◽  
Ice Water

BackgroundPreanalytic factors such as time and temperature can have significant effects on laboratory test results. For example, ammonium concentration will increase 31% in blood samples stored at room temperature for 30 min before centrifugation. To reduce preanalytic error, blood samples may be placed in precooled tubes and chilled on ice or in ice water baths; however, the effectiveness of these modalities in cooling blood samples has not been formally evaluated. The purpose of this study was to evaluate the effectiveness of various cooling modalities on reducing temperature of EDTA whole blood samples.MethodsPooled samples of canine EDTA whole blood were divided into two aliquots. Saline was added to one aliquot to produce a packed cell volume (PCV) of 40% and to the second aliquot to produce a PCV of 20% (simulated anemia). Thirty samples from each aliquot were warmed to 37.7 °C and cooled in 2 ml allotments under one of three conditions: in ice, in ice after transfer to a precooled tube, or in an ice water bath. Temperature of each sample was recorded at one minute intervals for 15 min.ResultsWithin treatment conditions, sample PCV had no significant effect on cooling. Cooling in ice water was significantly faster than cooling in ice only or transferring the sample to a precooled tube and cooling it on ice. Mean temperature of samples cooled in ice water was significantly lower at 15 min than mean temperatures of those cooled in ice, whether or not the tube was precooled. By 4 min, samples cooled in an ice water bath had reached mean temperatures less than 4 °C (refrigeration temperature), while samples cooled in other conditions remained above 4.0 °C for at least 11 min. For samples with a PCV of 40%, precooling the tube had no significant effect on rate of cooling on ice. For samples with a PCV of 20%, transfer to a precooled tube resulted in a significantly faster rate of cooling than direct placement of the warmed tube onto ice.DiscussionCanine EDTA whole blood samples cool most rapidly and to a greater degree when placed in an ice-water bath rather than in ice. Samples stored on ice water can rapidly drop below normal refrigeration temperatures; this should be taken into consideration when using this cooling modality.

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