Abstract

Introduction: Recombinant human erythropoietin (rHuEPO) administration studies involving transcriptomic approaches have demonstrated a gene expression signature that could aid blood doping detection. However, current anti-doping testing does not involve collecting whole blood into tubes with RNA preservative. This study investigated if whole blood in long-term storage and whole blood left over from standard hematological testing in short-term storage could be used for transcriptomic analysis despite lacking RNA preservation. Methods: Whole blood samples were collected from twelve and fourteen healthy nonathletic males, for long-term and short-term storage experiments. Long-term storage involved whole blood collected into Tempus™ tubes and K2EDTA tubes and subjected to long-term (i.e., ‒80°C) storage and RNA extracted. Short-term storage involved whole blood collected into K2EDTA tubes and stored at 4°C for 6‒48 h and then incubated at room temperature for 1 and 2 h prior to addition of RNA preservative. RNA quantity, purity, and integrity were analyzed in addition to RNA-Seq using the MGI DNBSEQ-G400 on RNA from both the short- and long-term storage studies. Genes presenting a fold change (FC) of >1.1 or < ‒1.1 with p ≤ 0.05 for each comparison were considered differentially expressed. Microarray analysis using the Affymetrix GeneChip® Human Transcriptome 2.0 Array was additionally conducted on RNA from the short-term study with a false discovery ratio (FDR) of ≤0.05 and an FC of >1.1 or < ‒1.1 applied to identify differentially expressed genes. Results: RNA quantity, purity, and integrity from whole blood subjected to short- and long-term storage were sufficient for gene expression analysis. Long-term storage: when comparing blood tubes with and without RNA preservation 4,058 transcripts (6% of coding and non-coding transcripts) were differentially expressed using microarray and 658 genes (3.4% of mapped genes) were differentially expressed using RNA-Seq. Short-term storage: mean RNA integrity and yield were not significantly different at any of the time points. RNA-Seq analysis revealed a very small number of differentially expressed genes (70 or 1.37% of mapped genes) when comparing samples stored between 6 and 48 h without RNA preservative. None of the genes previously identified in rHuEPO administration studies were differently expressed in either long- or short-term storage experiments. Conclusion: RNA quantity, purity, and integrity were not significantly compromised from short- or long-term storage in blood storage tubes lacking RNA stabilization, indicating that transcriptomic analysis could be conducted using anti-doping samples collected or biobanked without RNA preservation.

Highlights

  • Recombinant human erythropoietin administration studies involving transcriptomic approaches have demonstrated a gene expression signature that could aid blood doping detection

  • The long-term storage study was designed considering the following: 1) the ideal situation (RNA collection/extraction according to manufacturer instructions) as a reference and 2) the real situation that is faced by anti-doping biobanks, where whole blood samples are stored in tubes without an RNA stabilization reagent (e.g., K2EDTA tubes)

  • There were no statistical differences for the mean RIN of groups within the ideal situation

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Summary

Introduction

Recombinant human erythropoietin (rHuEPO) administration studies involving transcriptomic approaches have demonstrated a gene expression signature that could aid blood doping detection. The efficiency of the current system remains inadequate as the number of true doping cases vastly exceeds those detected (de Hon et al, 2014; Faiss et al, 2020). In response to this situation and to take advantage of advances in analytical techniques over the last few decades, WADA introduced the capacity for antidoping samples to be stored for the long term (initially 8 years, but currently 10 years) for retrospective re-analysis as new technologies and approaches emerge (Kuuranne and Saugy, 2016). Thereby, it has been argued that in addition to increasing preOlympic out-of-competition testing to avoid retrospective analysis to identify doping in the first place, long-term sample storage should continue and incorporate novel and potentially complementary technologies/sample matrices (Kolliari-Turner et al, 2021)

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