Optimized protocol for the extraction of RNA and DNA from frozen whole blood sample stored in a single EDTA tube.
Author(s): Yamagata H, Kobayashi A, Tsunedomi R, Seki T, Kobayashi M, Hagiwara K, Chen C, Uchida S, Okada G, Fuchikami M, Kamishikiryo T, Iga JI, Numata S, Kinoshita M, Kato TA, Hashimoto R, Nagano H, Okamoto Y, Ueno S, Ohmori T, Nakagawa S
Publication: Sci Rep, 2021, Vol. 11, Page 17075
PubMed ID: 34426633 PubMed Review Paper? No
Purpose of Paper
This paper compared RNA and DNA yields, purity and integrity when blood specimens were thawed by three different methods and when RNA extraction was performed with two different kits. The authors also compared fresh and frozen stored blood were also compared with respect to RNA and DNA yield, RNA integrity number (RIN), and variability in expression of housekeeping genes.
Conclusion of Paper
RNA yields were significantly higher regardless of thaw method when extraction was with the NucleoSpin rather than PAXgene kit. RNA yields were highest when blood was thawed on ice for 2 h, but DNA yields were highest when blood was thawed in room temperature aluminum blocks for 18 min. While RNA purity, assessed by A260/280 and A260/230 ratios, was unaffected by blood thaw method, DNA A260/280 and A260/230 ratios were significantly higher when blood was thawed in aluminum blocks at room temperature rather than on ice. Importantly, thawing blood in room temperature aluminum blocks for 18 min yielded RNA with a higher RIN and more amplifiable copies of GAPDH and RPS18 levels than when thawed at 37°C for 5 min, but thaw temperature did not have an obvious effect on DNA banding in electrophoresis. Finally, DNA and RNA yields were affected by the duration of frozen storage, as specimens stored for 6.1 ± 1.7 years at -80°C yielded more RNA and less DNA than fresh specimens, but the volume of tubes used for long term storage was also larger. There was no correlation between RNA yield or RIN and storage duration, but less variability in the expression of 5 of 23 housekeeping genes was observed when RNA was isolated from blood stored at -80°C for long durations in comparison to fresh blood.
Studies
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Study Purpose
In this study RNA yield, purity, and integrity were compared following RNA extraction from blood specimens thawed on ice, at room temperature, or at 37°C; two different RNA extraction kits were also compared. The authors also compared RNA yield, integrity, and variability in expression of housekeeping genes between RNA extracted from fresh blood and from blood stored frozen for years. Blood was collected from seven patients in a depression study into sodium EDTA and PAXgene tubes. RNA was extracted directly from PAXgene blood using the PAXgene RNA kit. EDTA blood was stored at -20°C overnight before transfer to -80°C for 48-72h. Aliquots were thawed in a 37°C water bath for 5 min, in a room temperature aluminum block for 18 min, or on ice for 2 h. RNA was extracted from thawed blood using either the PAXgene or NucleoSpin kit. RNA yield and purity were assessed by spectrophotometer. RNA integrity was evaluated using an Agilent RNA 6000 kit with a bioanalyzer and by real-time PCR amplification of GAPDH and RPS18. To investigate effects of long-term storage, thirty fresh blood specimens (from a depression study) and ten archival EDTA blood specimens (diagnosis not specified, stored 6.1 ± 1.7 years) that were thawed in room temperature aluminum blocks were compared; RNA from all specimens was isolated with the NucleoSpin kit. Libraries were constructed from RNA in the long-term storage experiment using the TruSeq Stranded Total RNA with Ribo-Zero Gold LT sample prep kit and pair-end sequenced on a NextSeq 500.
Summary of Findings:
RNA yields were significantly higher regardless of thaw method when extraction was with NucleoSpin rather than the PAXgene kit (P=1.4 x 10-7). Among specimens extracted using Nucleospin, RNA yields were lower when blood was thawed at 37°C for 5 min rather than on ice for 2 h (5.83 ± 2.31 versus 6.69 ± 2.04 μg/mL of blood, P=0.014); however, RNA yields were comparable when blood was thawed for 18 min in a room temperature aluminum block or on ice for 2 h (6.23 ± 2.29 versus 6.69 ± 2.04 μg/mL of blood, P=0.26). Overall, RINs were higher for specimens thawed in a room temperature aluminum block than in a 37°C water bath (P=0.044), but the difference between groups was not significant when only specimens extracted with NucleoSpin were considered. GAPDH and RPS18 levels quantified by real time RT-PCR did not differ among thaw conditions when RNA was isolated by Nucleospin; but when RNA was isolated with the PAXgene kit GAPDH and RPS18 levels were lower in blood specimens thawed at 37°C compared to specimens thawed in a room temperature aluminum block (P=0.036 and P=0.033, respectively) or fresh blood specimens (P=0.0001 and P=8.9 x 10-4, respectively). When RNA was isolated by NucleoSpin, A260/280 and A260/230 ratios were comparable for all three thaw methods. Finally, greater RNA yields were observed among specimens stored frozen for 6.1 ± 1.7 years compared to specimens that underwent short-term storage (both were thawed in aluminum blocks at room temperature and extracted with NucleoSpin) (6.69 ± 2.04 versus 6.23 ± 2.29 versus μg/mL, P=0.006), but the tube volume was also larger. There was no correlation between RNA yield or RIN and storage duration. Interestingly there was also less variability in the expression of 23 housekeeping genes when the blood experienced long term frozen storage compared fresh specimens, with significantly lower variability found for five of the genes.
Biospecimens
Preservative Types
- PAXgene
- Frozen
Diagnoses:
- Not specified
- Depression
Platform:
Analyte Technology Platform RNA Next generation sequencing RNA Automated electrophoresis/Bioanalyzer RNA Real-time qRT-PCR RNA Spectrophotometry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Thaw duration 5 min (37°C)
2 h (on ice)
18 min (room temperature aluminum block)
Storage Thaw temperature/condition 37°C water bath
Room temperature aluminum block
On ice
Analyte Extraction and Purification Analyte isolation method PAXgene tube blood RNA kit
Real-time qRT-PCR Specific Targeted nucleic acid GAPDH
RPL18
Storage Storage duration 0 h
6.1 ± 1.7 years
Biospecimen Preservation Type of fixation/preservation Frozen
PAXgene
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Study Purpose
This paper compared DNA yield, purity, and integrity among blood specimens thawed by three different methods. The authors also compared DNA yields of fresh blood and blood stored frozen for years. Blood was collected from six patients in a depression study into sodium EDTA and PAXgene tubes. DNA was extracted directly from PAXgene blood using the PAXgene Blood kit. EDTA blood was stored at -20°C overnight before transfer to -80°C for 48-72 h. Frozen aliquots were thawed in a 37°C water bath for 5 min, in a room temperature aluminum block for 18 min, or on ice for 2 h. DNA was extracted from thawed blood using the QIAamp DNA kit and quantified by spectrophotometer. DNA integrity was also assessed by electrophoresis. To investigate effects of long-term storage, DNA was extracted from thirty fresh blood specimens collected during a depression study and ten archival EDTA blood specimens (diagnosis not specified, stored 6.1 ± 1.7 years) that were thawed in room temperature aluminum blocks.
Summary of Findings:
DNA yields were significantly higher when blood was thawed in the room temperature aluminum block than on ice (25.19 ± 4.99 μg/mL versus 21.40 ± 6.37 μg/mL, P=0.021) or at 37°C (25.19 ± 4.99 μg/mL versus 19.40 ± 7.48 μg/mL, P=0.023). While A260/280 and A260/230 ratios were significantly higher when blood specimens were thawed at room temperature than on ice (1.91 ± 0.03 versus 1.90 ± 0.03, P=0.002), the authors state the difference was not meaningful. Importantly, no smears were observed among DNA samples when evaluated by gel electrophoresis. DNA yield was lower from blood specimens stored for 6.1 ± 1.7 years (18.15 ± 4.46 versus 25.19 ± 4.99 μg/mL) compared to fresh specimens, but statistical significance was not assessed.
Biospecimens
Preservative Types
- PAXgene
- Frozen
Diagnoses:
- Not specified
- Depression
Platform:
Analyte Technology Platform DNA Spectrophotometry DNA Electrophoresis Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Thaw duration 5 min (37°C)
2 h (on ice)
18 min (room temperature aluminum block)
Storage Thaw temperature/condition 37°C water bath
Room temperature aluminum block
On ice
Storage Storage duration 0 h
6.1 ± 1.7 years
Biospecimen Preservation Type of fixation/preservation PAXgene
Frozen