Extraction of high quality and high yield RNA from frozen EDTA blood.pdf
1 Vol.:(0123456789) Scientific Reports |         (2024) 14:8628   | https://doi.org/10.1038/s41598-024-58576-9 www.nature.com/scientificreports Extraction of high quality and high  yield RNA from frozen EDTA blood Long T. Nguyen *, Carol A. Pollock  & Sonia Saad  Peripheral blood RNA profiling, which can reveal systemic changes in gene expression and immune  responses to disease onset and progression, is a powerful tool for diagnosis and biomarker discovery.  This technique usually requires high quality RNA, which is only obtainable from fresh blood, or  frozen blood that has been collected in special RNA-stabilisation systems. The current study aimed  to develop a novel protocol to extract high quality RNA from frozen blood that had been collected  in the conventional EDTA tubes. We determined that thawing EDTA blood in the presence of cell  lysis/RNA stabilisation buffers (Paxgene or Nucleospin) significantly improved RNA quality (RIN)  from below 5 to above 7, which to date has not been shown possible. The EDTA-Nucleospin protocol  resulted in 5 times higher yield than the EDTA-Paxgene-PreAnalytix method. The average RIN and  mRNA expression levels of five different genes including 18 s, ACTB, MCP1, TNFa and TXNIP using this  protocol were also indifferent to those from Paxgene blood, suggesting similar RNA quality and blood  transcriptome. Moreover, the protocol allows DNA to be extracted simultaneously. In conclusion, we  have developed a practical and efficient protocol to extract high quality, high yield RNA from frozen  EDTA blood. Blood is a near unlimited source of biological materials to be used for disease diagnosis and monitoring. Blood is  routinely collected from both healthy individuals and patients due to the minimal invasiveness of the procedure.  Additionally, in research, blood-derived proteins, metabolites, DNA and RNA can all be used for biomarker  discovery and mechanistic investigation of different diseases. For example, blood RNA profiling has been used to  identify candidate biomarkers for classification and prognostication of diabetes and ­complications1–4, pulmonary  ­disease5, cardiovascular ­disease6,7, neurological ­diseases8,9, and ­cancer10,11. However, RNA is highly susceptible to  degradation due to its relatively unstable structure and the ubiquitous presence of RNases in the environment.  Suboptimal collection and storage conditions of blood can lead to haemolysis and release of RNases from red  blood cells, resulting in RNA degradation. Degraded RNA is generally not recommended for library construction  in next generation sequencing, especially when coding regions are of primary interest. Ethylenediaminetetraacetic acid (EDTA)-coated tubes are the conventional containers for blood collection,  which although can be used to extract intact RNA from fresh blood, does not contain RNA stabilising chemicals  to protect RNA from degradation during freezing and thawing. It is well-known that RNA extracted from frozen  EDTA blood not only has a low yield but is also highly ­fragmented12,13. In most cases, blood is collected at the  clinic but processed centrally later elsewhere. Freezing can temporarily deactivate RNases but freeze–thaw cycles  can result in uncontrolled haemolysis and release of RNases, making RNA degradation inevitable. As such, legacy  blood samples that were collected in EDTA tubes are generally deemed not suitable for transcriptional profiling. RNA stabilisation systems such as Paxgene (BD Biosciences) or Tempus (Thermofisher Scientific) are now  available to address the issue. In this method, whole blood needs to be mixed with specific buffers at the time of  collection to lyse blood cells and inhibit RNases in a well-controlled manner. RNA is precipitated from the solu- tion in these buffers and therefore preserved intact for later purification. However, there have been reports show- ing that RNA extracted from Paxgene tubes contained DNA contamination or inefficient in PCR ­reaction13,14.  Moreover, as these kits are expensive and incompatible with other biochemical assays, they are usually not  routinely stocked for clinical use unless being specified as part of a clinical trial. This means to extract intact  RNA for transcriptional profiling, prospective human studies are generally required, with blood being drawn  for storage in tubes suitable for RNA extraction which involves extensive commitment and inevitable cost that  otherwise could be avoided. The recruitment of new patient cohorts also means that the use of historic biobanks  are not optimally utilised and current and past data cannot be correlated, thus limiting data interpretation. For standard RNA-seq library preparation, the recommended RNA amount and RNA integrity (RIN) is  500 ng and 7–8 respectively. Higher RINs mean that RNA is less degraded. RNA extracted from frozen EDTA  blood using traditional methods (e.g. Trizol) tends to have RIN of < ­412,15,16. Beekman and colleagues attempted  OPEN Renal Medicine, Kolling Institute, University of Sydney, Camperdown, Australia. *email: long.t.nguyen@sydney. edu.au 2 Vol:.(1234567890) Scientific Reports |         (2024) 14:8628  |  https://doi.org/10.1038/s41598-024-58576-9 www.nature.com/scientificreports/ to overcome this problem by transferring EDTA blood to PAXgene tubes followed by RNA extraction using a  PAXgene-compatible kit. This approach was shown to increase RNA yield and integrity (RIN = 6)12, which may  be acceptable for certain applications such as ­microarray12. However, as the impact of suboptimal RIN on RNA  sequencing is unclear, RNA with RIN > 7 is still requested by Illunima and most sequencing service providers,  particularly when mRNA is of the primary interest. Several groups have attempted to improve the quality of EDTA blood-derived RNA to meet mRNA-Seq  ­standards15,16. However, RIN = 6 appears to be the highest achievable value and thus still remain suboptimal.  The ability to extract high-quality RNA from frozen EDTA blood will allow researchers to simplify and stand- ardise the sample preparation procedure to achieve the highest efficiency and reproducibility in RNA biomarker  research. Legacy cryopreserved blood samples across the world can be used and data generated from the same  samples/cohort can be correlated to gain novel insights into different diseases and therapeutic effects. This study  aimed to compare different RNA extraction protocols to identify the determining factors to extract high quality  RNA from frozen EDTA blood for mRNA-seq. Results The effects of incubation and extraction kits in the EDTA‑to‑PAXgene transfer method on  blood RNA quantity and quality First, we assessed the impact of using different kits to extract RNA from EDTA blood that was transferred into  Paxgene tubes before extraction. As shown in Table 1, EDTA-Paxgene-PreAnalytix protocol showed a 36%  increase in yield compared to the PAXgene PreAnalytix (PP) control method. However, RIN was approximately  halved (P < 0.05) and A260/230 was significantly lower (P < 0.01), suggesting the expected poor quality. If the  Magmax extraction kit was used instead of PreAnalytix, we found no change in yield, a slightly less drop in RIN  (P < 0.01) but a much more significant reduction in A260/230 (P < 0.01). Next, we questioned if varying sample incubation time before RNA extraction would make an impact on  RNA quantity and quality. The result showed that there were no improvements in any of the measurements  when the blood was incubated for either 2 h or overnight before extraction. Refreezing the blood in − 80 °C also  had no effect. The effect of cell lysis/RNA stabilisation buffer As RNA integrity remained low despite blood was transferred to Paxgene system after thawing, we tried to  address the key question as to whether RNA degradation occurred during cryopreservation or during the thawing  process. If degradation had already occurred during cryopreservation, then attempts to improve RNA integrity  would not be fruitful. By adding Paxgene additives to EDTA tubes during thawing instead of vice versus, RIN  was significantly improved to 7.3 ± 0.14, while RNA yield remained similar (P < 0.01, Table 2). To confirm that this effect is not specifically attributed to Paxgene additives but to the reverse addition of cell  lysis/RNA stabilisation buffer, we tested the Nucleospin blood RNA extraction from Macherey–Nagel. Similar to  the EDTA-Paxgene reverse transferring method followed by PreAnalytix extraction kit, adding Nucleospin lysis  buffer to EDTA blood before thawing resulted in a significantly improved RIN of 8 ± 0.21 (P < 0.01). At the same  time, RNA yield was also 5 times higher than EDTA-PP method (P < 0.01, Table 2). This protocol is referred to  as EDTA-mixed thawing-Nucleospin (EmN). Table 1.   RNA extraction following blood transfer from EDTA to Paxgene tubes. EDTA blood was thawed on  ice for at least 2 h before transferring to Paxgene tubes. Data are expressed as Mean ± SEM. N = 2. *P < 0.05,  **P < 0.01. Protocol Yield (μg RNA/ml blood) % PP-Ctrl RIN 260/280 260/230 Paxgene Ctrl PreAnalytix 1.00 ± 0.31 100.00 8.00 ± 0.2 2.14 ± 0.06 1.54 ± 0.04 EDTA blood transfer to  Paxgene tube Kit PreAnalytix 1.38 ± 0.48 136.76 ± 5.74* 4.00 ± 0.3** 2.15 ± 0.08 0.94 ± 0.11* Magmax 1.04 ± 0.23 107.10 ± 9.56 4.55 ± 0.25** 2.15 ± 0.05 0.39 ± 0.14** Incubation 2 h 0.85 ± 0.03 123.07 ± 3.65 4.87 ± 0.30** 2.08 ± 0.01 0.59 ± 0.21** overnight 1.37 ± 0.22 104.83 ± 17.31 3.97 ± 0.15** 2.22 ± 0.01 0.57 ± 0.11** Refreeze No 1.21 ± 0.24 107.27 ± 11.98 4.28 ± 0.23** 2.15 ± 0.04 0.66 ± 0.17** Yes 0.90 ± 0.05 83.60 ± 6.47 4.70 ± 0.57** 2.16 ± 0.06 0.42 ± 0.03** Table 2.   The effect of lysis buffers in Paxgene and Nucleospin kits on blood RNA degradation. Data are  expressed as Mean ± SEM. N = 2. **P < 0.01. Blood tube Pretreatment Incubation Extraction %PP-Ctrl RIN EDTA Thaw on ice then transfer to Paxgene tube RT (2 h) Pre-AnalytiX 136.76 ± 5.74 4.00 ± 0.3 EDTA Add Paxgene solution to EDTA tube then thaw on ice RT (2 h) Pre-AnalytiX 148.77 ± 33.79 7.30 ± 0.14** EDTA Add Nucleospin lysis buffer then thaw on ice RT (15 min) Nucleospin 649.77 ± 72.51** 8.00 ± 0.21** 5 Vol.:(0123456789) Scientific Reports |         (2024) 14:8628  |  https://doi.org/10.1038/s41598-024-58576-9 www.nature.com/scientificreports/ worst case, certain mRNA can be completely lost or degraded during upstream workflow steps, which increases  the risk of false-negative results. As such, regardless of whether total RNA-seq or mRNA-seq is used, a high RIN  is always preferable. Moreover, compared to total RNA-seq, library preparation can be performed with smaller  sample sizes for mRNA-Seq and sequencing depth can be increased, which significantly improves sequencing  efficiency and reduces cost. In comparing the Paxgene/PreAnalytix control method and EDTA-Nucleospin, the latter has been shown to  improve RNA yield by 5 ­times16. The same result was achieved in the current study, suggesting that Nucleospin  is indeed much more efficient in RNA recovery. This is critical when the amount of blood for RNA extraction  is limited, which is generally the case if blood is historically stored from clinical trial participants. In addition,  we found Nucleospin protocol is much simpler. It has only 8 steps and takes ~ 45 min to complete, compared to  PreAnalytix kit which consists of 21 steps and can require up to 2 h of processing time. Regarding RNA quality  and expression, our improved protocol showed equivalent RIN and identical expression levels of 5 reference  genes, suggesting data of the two methods are likely comparable, although whole transcriptomic sequencing is  required to fully assess similarity of the two expression profiles. A potential drawback of the mixing-before-thawing method is that blood in a whole EDTA tube generally  needed to be entirely ­thawed16, therefore cannot be saved for other purposes. In most cases this should not be  a problem because most blood-based assays such as ELISA and metabolite measurement can only be done on  serum/plasma, which requires separation from fresh blood before cryopreservation. Frozen whole blood is pri- marily used for DNA extraction and as we demonstrated in this study, it is also possible to concurrently extract  RNA and DNA after the blood is treated with cell lysis buffer from Nucleospin blood RNA kit. A proportion of  the lysed blood (up to 1.3 ml) will continue to be used for RNA extraction by the same kit, while the rest can  be used for DNA extraction using Nucleospin blood DNA kit. This dual extraction method showed sufficient  yield for next-gene sequencing of both RNA and DNA. Although A260/230 of the extracted DNA was relatively  low, this could be further addressed by running the samples through Bio-spin columns multiple times until a  desirable A260/230 is achieved. In summary, we have optimised a protocol to extract high quality and quantity RNA from EDTA tubes to  study gene expression. In addition, Nucleospin blood RNA and DNA kits can be used together to isolate both  RNA and DNA from the same extraction. Should the new method be adopted, all legacy EDTA blood can be  utilised for gene expression profiling and future studies no longer need to rely on expensive RNA stabilising  systems for blood storage. Methods Patients Participants of either sex aged between 18 and 75 years of age were included. Informed consent was obtained  from all participants involved in this study. The study was carried out in accordance with relevant guidelines  and regulations was approved by the human Ethics committee at Royal North Shore Hospital (Ref: HREC/17/ HAWKE/471). Blood from a volunteer (~ 10 ml/collection) was used to optimise RNA quantity and quality.  Then a cohort of six healthy individuals were recruited to compare the novel method with the standard protocol  using PAXgene system. Sample collection and storage For each participant, blood was collected by a clinical nurse in one PAXgene tube (2.5 ml) and two EDTA tubes  (4 ml/tube) respectively. PAXgene tubes (Becton–Dickinson) were mixed thoroughly and stored in − 20 °C over- night followed by long-term storage in − 80 °C freezer. Blood collected in EDTA tubes (Sarstedt) were directly  stored in − 80 °C freezer. Samples were kept at − 80 °C for 2–3 months until RNA extraction. RNA extraction strategies For RNA extraction from original Paxgene tubes, samples were thawed on ice for at least 2 h and PAXgene Blood  RNA Kit (PreAnalytix, Hombrechtikon, Switzerland) was used to extract blood RNA according to the manu- facturer’s manual. This method is referred to as Paxgene Control or PP. For EDTA tubes, we had two strategies  to optimise the RNA quantity and quality. The first was to replicate and optimise a reference protocol based on  transferring EDTA blood to PAXgene tubes before extraction. Several conditions were then tested including (1)  Different extraction kits: PAXgene Blood RNA Kit (PreAnalytix, Hombrechtikon, Switzerland) versus MagMAX  blood RNA Isolation Kit for PAXgene (Thermofisher Scientific, MA, USA), (2) different post-transfer incubation  time: 2 h versus overnight, and (3) same day extraction vs refreezing the samples for later extraction. The second  strategy was to optimise the Nucleospin blood RNA extraction kit (Macherey–Nagel, Westfalen, Germany),  which was primarily meant to be used for fresh blood but has been shown to produce a much higher yield of  RNA compared to other protocols using frozen ­blood15,16. DNA extraction 4 ml of blood was thawed using lysis buffer in Nucleospin blood RNA kit then 2.0 ml of blood was moved into  a new centrifuge tube for DNA isolation. Blood DNA was extracted using Nucleospin Blood DNA Midi Kit  according to the manufacturer’s instruction, followed by salt clearance using Micro Bio-Spin™ P-30 Gel Columns,  Tris Buffer (Bio-rad, CA, USA). RNA/DNA quantity and quality check RNA concentration and quality were measured by TapeStation 4200 (Agilent, CA, USA). DNA concentration  was measured by Nanodrop 100. A260/A230 and A280/260 were measured using Nanodrop. 6 Vol:.(1234567890) Scientific Reports |         (2024) 14:8628  |  https://doi.org/10.1038/s41598-024-58576-9 www.nature.com/scientificreports/ Real‑time qPCR 500 ng of RNA from each sample was used for cDNA synthesis using iScript™ gDNA Clear cDNA Synthesis  Kit (Bio-rad, CA, USA) and RT-PCR was run using iTaq™ Universal SYBR® Green Supermix according to the  manufacturer’s instructions. In order to compare the mRNA expression profiles of the blood processed by the  reference protocol and the novel method, two reference genes including 18 s and β-actin (ACTB) were exam- ined. In addition, Monocyte Chemoattractant Protein 1 (MCP1), Tumour necrosis factor alpha (TNF-α) and  Thioredoxin-Interacting Protein (TXNIP) were tested as genes of interest as they are involved in inflammation  and diabetes (ref). Statistics For protocol optimisation, each condition was tested in duplicate on two different days using blood from the same  volunteer, and data are expressed as Mean ± SEM. For validation, a human cohort of n = 6 was used and data are  expressed as Mean ± SD. Unpaired t-tests were used for each comparison and P < 0.05 was considered significant. Data availability The datasets generated during and/or analysed during the current study are available from the corresponding  author on reasonable request. Received: 2 January 2024; Accepted: 1 April 2024 References 	 1.	 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Title: Extraction of high quality and high yield RNA from frozen EDTA blood ​


Authors: Long T. Nguyen, Carol A. Pollock, Sonia Saad, H. Yamagata, et al. ​
Publisher: Springer Nature ​​​​
Date: 2021
Abstract: The study developed a novel protocol for extracting high-quality RNA from frozen EDTA blood, enabling RNA profiling and biomarker discovery. ​ The protocol significantly improved RNA quality, achieving a RIN above 7, and resulted in 5 times higher RNA yield compared to existing methods. ​ It also allowed for simultaneous DNA extraction, providing a practical approach for gene expression profiling and biomarker research.