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

ABSTRACT: Cryopreservation 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.

SUBMITTER: Yamagata H

PROVIDER: S-EPMC8382694 | biostudies-literature | 2021 Aug

REPOSITORIES: biostudies-literature

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

Yamagata Hirotaka H Kobayashi Ayumi A Tsunedomi Ryouichi R Seki Tomoe T Kobayashi Masaaki M Hagiwara Kosuke K Chen Chong C Uchida Shusaku S Okada Go G Fuchikami Manabu M Kamishikiryo Toshiharu T Iga Jun-Ichi JI Numata Shusuke S Kinoshita Makoto M Kato Takahiro A TA Hashimoto Ryota R Nagano Hiroaki H Okamoto Yasumasa Y Ueno Shuichi S Ohmori Tetsuro T Nakagawa Shin S

Scientific reports 20210823 1

Cryopreservation 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 ...[more]

PMID: 34426633

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Project description:BackgroundThe use of RBC lysate (RBC-Lys) eliminates the need for serum folate and hematocrit (Hct) measurement to calculate RBC folate. Information on the long-term frozen storage stability of RBC-Lys is missing.ObjectivesWe aimed to assess the comparability of RBC folate forms in whole-blood lysate (WB-Lys) and RBC-Lys and the folate stability in both matrices.MethodsWe prepared conventional WB-Lys (1:11 dilution with 1% ascorbic acid) and RBC-Lys (1:11 dilution of washed and saline-diluted RBCs with 1% ascorbic acid) from EDTA blood (n = 60 adult donors) and stored lysates at -70°C until analysis at baseline (1 wk), 3, 6, 12, and 24 mo. Before analysis by HPLC-tandem MS, we incubated the WB-Lys (4 h at 37°C) and treated the RBC-Lys with human recombinant γ-glutamyl hydrolase for folate polyglutamate deconjugation. We analyzed RBC-Lys samples for hemoglobin (Hb) (same aliquot) to normalize for the preanalytical dilution; Hb-folate was converted to RBC folate for each folate form using the mean corpuscular Hb concentration. We analyzed Hct as well as folate forms in matching serum samples for traditional RBC folate calculation. We conducted descriptive data analyses (correlation, Bland-Altman plot, Deming regression).ResultsAt baseline, results for RBC folate forms derived from WB-Lys compared with RBC-Lys samples showed excellent correlation (Pearson r ≥ 0.97). Mean ± SD concentrations compared well for total folate (WB-Lys: 886 ± 255 compared with RBC-Lys: 899 ± 271 nmol/L), 5-methyltetrahydrofolate (WB-Lys: 831 ± 258 compared with RBC-Lys: 843 ± 276 nmol/L), and nonmethyl folate (WB-Lys: 53.3 ± 74.4 compared with RBC-Lys: 52.9 ± 70.7 nmol/L), but were 17% higher in RBC-Lys for pyrazino-s-triazine derivative of 4α-hydroxy-5-CH3-H4folate (MeFox) (WB-Lys: 147 ± 44.1 compared with RBC-Lys: 172 ± 53.5 nmol/L). Frozen storage of WB-Lys and RBC-Lys samples for ≤24 mo showed ≤5%, ≤5%, ≤13%, and ≤11% change in total folate, 5-methyltetrahydrofolate, nonmethyl folate, and MeFox, respectively.ConclusionsErythrocyte folate forms appear to be stable in RBC-Lys samples stored frozen at -70°C for ≤2 y. The relatively small changes in folate concentrations over time were comparable between RBC-Lys and conventionally prepared WB-Lys samples.

Project description: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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Optimized protocol for the extraction of RNA and DNA from frozen whole blood sample stored in a single EDTA tube.

Publications

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

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