By Pooja Toshniwal PahariaReviewed by Lexie CornerMay 28 2025A recent study in Nature Communications investigated how pre-analytical factors affect the transcriptome of blood-based extracellular RNA (exRNA, or cell-free RNA) biomarkers.
The researchers established guidelines for blood collection tube type, RNA purification methods, and processing time to help optimize exRNA profiling. These recommendations aim to support both researchers planning exRNA studies and manufacturers evaluating new products.
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Background
Biomarkers are important in early diagnosis, treatment monitoring, and disease risk prediction. Liquid biopsies, derived from biofluids, offer a less invasive method for ongoing health assessment.
While cell-free DNA is already widely used for mutation detection and non-invasive prenatal testing (NIPT), exRNA is a more recently explored biomarker. Its use requires standardized protocols for sample collection, processing, and analysis to minimize technical variability.
Plasma and serum are the most studied biofluids in this context. Pre-analytical factors—including the choice of blood collection tube, RNA extraction method, and processing delay—can influence the quantity and quality of exRNA. However, the extent of these effects on transcriptome-wide exRNA profiles remains unclear.
About the Study
In the exRNA Quality Control (exRNAQC) study, researchers evaluated how various pre-analytical variables impact exRNA profiling. The team characterized the transcriptomes of messenger RNA (mRNA) and microRNA (miRNA), using methods that removed genomic DNA to prevent contamination.
The study assessed:
- 10 types of blood collection tubes, including five standard tubes and five designed to preserve nucleic acids.
- Eight RNA purification techniques.
- Three processing time intervals.
The analysis included 456 extracellular transcriptomes from 20 donors (five male, fifteen female, aged 27 to 54).
Preservation tubes were designed to stabilize nucleic acids, allowing for longer processing times. Processing intervals for standard tubes ranged from 4 to 16 hours, while for preservation tubes, processing occurred one to two weeks after collection.
The RNA purification techniques differed in eluate volume. High-volume methods included Maxwell, mirVana, and Norgen; low-volume methods included miRNeasy, miRNeasy Advanced, and QIAamp. Additional techniques included NucleoSpin and MagNA.
Performance metrics for RNA purification included:
- Absolute counts of mRNA and miRNA (sensitivity)
- Eluate RNA concentration
- Purification efficiency
- Count thresholds
- Replicate variability
- Read duplication rates
- Transcriptome coverage
Gene set enrichment analysis (GSEA) revealed changes in mRNA expression related to processing time in both standard and preservation tubes. Computational analysis also showed time-dependent shifts in RNA contributions from various immune cell types, depending on the tube used.
Results
Higher input volumes and lower eluate volumes generally led to better RNA yield, lower variability, and more complete transcriptome coverage. An exception was observed with the miRNeasy Advanced method, which had lower performance compared to other techniques using similar input volumes.
For miRNA, the NucleoSpin, Norgen, and QIAamp methods yielded fewer microRNAs despite high input volumes. QIAamp demonstrated the lowest read duplication rate (82 % using 4 mL plasma), producing six times more non-duplicated reads than other methods.
Preservation tubes showed signs of hemolysis that were not observed in standard tubes. RNA concentrations remained stable in standard tubes but decreased significantly in preservation tubes—up to fourfold in the case of RNA Streck.
Variability between replicates was low for both tube types. However, extended processing times increased gene expression variability (log2 fold-change), particularly in preservation tubes. Citrate and EDTA tubes showed stable RNA profiles after four hours, while only ACD-A and citrate tubes maintained consistent immune cell composition.
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Recommendations
Based on the exRNAQC study findings, researchers recommend collecting blood samples in citrate tubes and processing them within four hours. They advise against using preservation tubes such as DNA Streck, RNA Streck, Roche, PAXgene, and Biomatrica.
To maximize mRNA count while controlling for replicate variability, they recommend the QIAamp technique for large biofluid input volumes (≤4.0 mL) and miRNeasy or miRNeasy Advanced for small input volumes (≤0.60 mL). A separate evaluation for miRNA and mRNA is necessary.
Evaluating interactions between the pre-analytical variables is essential to determine the most suitable combination for exRNA research and application in biomarker studies.
Journal Reference
The exRNAQC Consortium. (2025). Blood collection tube and RNA purification method recommendations for extracellular RNA transcriptome profiling. Nat Commun. DOI: https://doi.org/10.1038/s41467-025-58607-7, https://www.nature.com/articles/s41467-025-58607-7
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