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Identification of RNA Markers in Red Blood Cells for Doping Control in Autologous Blood Transfusion

ABSTRACT: Identification of RNA Markers in Red Blood Cells for Doping Control in Autologous Blood Transfusion

PROVIDER: PRJNA847776 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR19612639_1.fastq.gz	Fastqsanger.gz
	SRR19612639_2.fastq.gz	Fastqsanger.gz
	SRR19612640_1.fastq.gz	Fastqsanger.gz
	SRR19612640_2.fastq.gz	Fastqsanger.gz
	SRR19612641_1.fastq.gz	Fastqsanger.gz

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Identification of RNA Markers in Red Blood Cells for Doping Control in Autologous Blood Transfusion

Project description:We conducted an animal experiment and used total RNA sequencing (RNA-Seq) to identify novel RNA markers to detect autologous blood transfusion (ABT) doping within red blood cells (RBCs) as a pilot study before human trials. The total RNA-Seq and bioinformatics were performed using rat RBCs that were stored 10- and 20-day periods with citrate–phosphate–dextrose solution with adenine (CPDA) at 4°C In the results, we identified three significant genes whose expression levels were drastically decreased according to the storage period. Our novel insights may allow future studies to develop a test method for ABT doping.

2022-07-14 | GSE205806 | GEO

In vivo detection of autologous blood doping

Project description:On the trail of blood doping – a microRNA-based biomarker signature to detect autologous blood transfusions in vivo

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Identification of RNA Markers in Red Blood Cells for Doping Control in Autologous Blood Transfusion.

Project description:The World Anti-Doping Agency (WADA) has prohibited the use of autologous blood transfusion (ABT) as a doping method by athletes. It is difficult to detect this doping method in laboratory tests, and a robust testing method has not yet been established. We conducted an animal experiment and used total RNA sequencing (RNA-Seq) to identify novel RNA markers to detect ABT doping within red blood cells (RBCs) as a pilot study before human trials. This study used whole blood samples from Wistar rats. The whole blood samples were mixed with a citrate-phosphate-dextrose solution with adenine (CPDA) and then stored in a refrigerator at 4 °C for 0 (control), 10, or 20 days. After each storage period, total RNA-Seq and bioinformatics were performed following RNA extraction and the purification of the RBCs. In the results, clear patterns of expression fluctuations were observed depending on the storage period, and it was found that there were large numbers of genes whose expression decreased in the 10- and 20-day periods compared to the control. Moreover, additional bioinformatic analysis identified three significant genes whose expression levels were drastically decreased according to the storage period. These results provide novel insights that may allow future studies to develop a testing method for ABT doping.

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Enhancement of red blood cell transfusion compatibility using CRISPR-mediated erythroblast gene editing

Project description:Regular blood transfusion is the cornerstone of care for patients with red blood cell (RBC) disorders such as thalassemia or sickle cell disease. With repeated transfusion, alloimmunisation often occurs due to incompatibility at the level of minor blood group antigens. We use CRISPR-mediated genome editing of an immortalised human erythroblast cell line (BEL-A) to generate multiple enucleation competent cell lines deficient in individual blood groups. Edits are combined to generate a single cell line deficient in multiple antigens responsible for the most common transfusion incompatibilities: ABO (Bombay phenotype), Rh (Rhnull), Kell (K0), Duffy (Duffynull), GPB (S- s- U-). These cells can be differentiated to generate deformable reticulocytes, illustrating the capacity for coexistence of multiple rare blood group antigen null phenotypes. This study provides the first proof-of-principle demonstration of combinatorial CRISPR-mediated blood group gene editing to generate customisable or multi-compatible RBCs for diagnostic reagents or recipients with complicated matching requirements.

2018-04-26 | PXD009291 | Pride

Integrating whole blood transcriptomic collection procedures into the current anti-doping testing system, including long-term storage and re-testing of anti-doping samples [Long-term]

Project description:Purpose: Recombinant human erythropoietin administration studies involving “omics” approaches have demonstrated a gene-expression signature that could aid detection of blood doping. However, current anti-doping testing does not involve blood collection into tubes with RNA preservative. This study investigated if whole blood in long-term storage could be used for transcriptomic analysis despite lacking RNA preservation. Methods: Whole blood samples were collected from thirteen male healthy individuals. Long-term storage: whole blood collected into Tempus™ tubes and K2EDTA tubes and subjected to long-term (i.e., −80°C) storage and RNA extracted. After storage, Tempus and K2EDTA tubes were thawed and extracted using Tempus™ Spin RNA Isolation Kit (Life Technologies, Carlsbad, CA, USA). Samples from seven subjects that presented higher RIN value (≥7) were selected for RNA_Seq analysis. Results: The experiment provided RNA quality and purity for gene expression analysis. Total of 19239 genes were mapped and the gene expression analysis showed that 658 genes were differentially expressed (which means 3.4% of mapped genes). With 269 being up-regulated and 389 down-regulated. None of the transcripts described in previous studies as biomarkers for blood doping (Durussel et al. 2016; Wang, Durussel, et al. 2017) were differently expressed. Conclusion: RNA quantity, purity and integrity was not significantly compromised from long-term storage in blood storage tubes lacking RNA stabilisation, indicating that transcriptomic/omics analysis could be conducted using anti-doping samples collected or biobanked without RNA preservation.

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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

Project description:Recombinant human erythropoietin administration studies involving transcriptomic approaches have demonstrated a gene-expression signature that could aid detection of blood doping. However, current anti-doping testing does not involve blood collection into tubes with RNA preservative. This study investigated if whole blood in long-term storage and whole blood leftover from standard haematological testing in short-term storage could be used for transcriptomic analysis despite lacking RNA preservative. Whole blood samples were collected from thirteen and fourteen healthy males, for long-term and short-term storage experiments. Long-term storage: whole blood collected into Tempus™ tubes and K2EDTA tubes and subjected to long-term (i.e., −80°C) storage and RNA extracted. After storage, K2EDTA tubes were thawed and extracted using GeneJET RNA Purification Kit (Thermo Fisher Scientific, Vilnius, Lithuania) or Tempus™ Spin RNA Isolation Kit (Life Technologies, Carlsbad, CA, USA). RNA quality and purity was sufficient for gene expression analysis. Principle Component Analysis of microarray and RNA-seq gene expression data for long-term storage: When comparing gene expression between blood tubes with and without RNA preservation, 6% (4058 transcripts) were differentially expressed. RNA quantity, purity and integrity was not significantly compromised from long-term storage in blood storage tubes lacking RNA preservative, indicating that transcriptomic analysis could be conducted using anti-doping samples collected or biobanked without RNA preservation.

2021-08-13 | GSE181970 | GEO

Integrating whole blood transcriptomic collection procedures into the current anti-doping testing system, including long-term storage and re-testing of anti-doping samples [Short-term]

Project description:Purpose: Recombinant human erythropoietin administration studies involving “omics” approaches have demonstrated a gene-expression signature that could aid detection of blood doping. However, current anti-doping testing does not involve blood collection into tubes with RNA preservative. This study investigated if whole blood in short-term storage could be used for transcriptomic analysis despite lacking RNA preservation. Methods: Whole blood samples were collected from fourteen male healthy individuals. Short-term storage: whole blood collected into K2EDTA tubes and subjected to short-term (i.e., at 4°C) storage for 6 hours, 12 hours, 24 hours and 48 hours. After storage, blood from K2EDTA tubes were transferred into Tempus™ Blood tubes, and then extracted using Tempus™ Spin RNA Isolation Kit (Life Technologies, Carlsbad, CA, USA). Samples from four subjects of each time point that presented higher RIN value (≥7) were selected for RNA_Seq analysis. Results: The experiment provided RNA quality and purity for gene expression analysis. Considering 6-hours storage as a reference group, the number of differentially expressed genes were 19, 45 and 70 in comparison to 12, 24 and 48-hours, respectively (which means 0.37, 0.88 and 1.37% of mapped genes). Of the 19 differentially expressed genes in the comparison 6 vs. 12-hours, 9 overlapped with the 45 in the comparison 12 vs. 24-hours. Furthermore, 40 of those 45 overlapped with the 70 differentially expressed in the comparison 6 vs. 48-hours. None of the transcripts described in previous studies (Durussel et al. 2016; Wang, Durussel, et al. 2017) were differently expressed. Conclusion: RNA quantity, purity and integrity was not significantly compromised from short-term storage in blood storage tubes lacking RNA stabilisation, indicating that transcriptomic/omics analysis could be conducted using anti-doping samples collected without RNA preservation.

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Proof of Gene Doping in a Mouse Model with a Human Erythropoietin Gene Transferred Using an Adenoviral Vector

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