Stabilization of cellular RNA in blood during storage at room temperature: a comparison of cell-free RNA BCT(®) with K3EDTA tubes.

BACKGROUND: Messenger RNA (mRNA) expression levels in blood cells are important in disease diagnosis, prognosis and biomarker discovery research. Accurate measurements of intracellular mRNA levels in blood cells depend upon several pre-analytical factors, including delays in RNA extraction from blood after phlebotomy. Dramatic changes in mRNA expression levels caused by delays in blood sample processing may render such samples unsuitable for gene expression analysis. OBJECTIVES: This study was conducted to evaluate a blood collection tube, cell-free RNA-BCT(®) (RNA-BCT), for its ability to stabilize mRNA expression level in blood cells post-phlebotomy using indicator mRNAs in reverse transcription quantitative real-time PCR (RT-qPCR) assays. METHODS: Blood samples from presumed healthy donors were drawn into both RNA-BCT and K3EDTA tubes and maintained at room temperature (18-22°C). The samples were processed to obtain white blood cells (WBCs) at days 0, 1, 2 and 3. Total cellular RNA was extracted from WBCs and mRNA concentrations were quantified by RT-qPCR for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), c-fos, and p53 transcripts. RESULTS: While blood cells isolated from K3EDTA tubes showed significant changes in cellular mRNA concentrations for GAPDH, c-fos, and p53, these mRNAs concentrations were stable in blood drawn into RNA-BCT. CONCLUSION: The reagent in the RNA-BCT device stabilizes cellular mRNA concentrations for GAPDH, c-fos and p53 for at least three days at room temperature.

A new blood collection device minimizes cellular DNA release during sample storage and shipping when compared to a standard device.

BACKGROUND: Cell-free DNA (cfDNA) circulating in blood is currently used for noninvasive diagnostic and prognostic tests. Minimizing background DNA is vital for detection of low abundance cfDNA. We investigated whether a new blood collection device could reduce background levels of genomic DNA (gDNA) in plasma compared to K(3) EDTA tubes, when subjected to conditions that may occur during sample storage and shipping. METHODS: Blood samples were drawn from healthy donors into K(3) EDTA and Cell-Free DNA™ BCT (BCT). To simulate shipping, samples were shaken or left unshaken. In a shipping study, samples were shipped or not shipped. To assess temperature variations, samples were incubated at 6°C, 22°C, and 37°C. In all cases, plasma was harvested by centrifugation and total plasma DNA (pDNA) assayed by quantitative real-time polymerase chain reaction (qPCR). RESULTS: Shaking and shipping blood in K(3) EDTA tubes showed significant increases in pDNA, whereas no change was seen in BCTs. Blood in K(3) EDTA tubes incubated at 6°C, 22°C, and 37°C showed increases in pDNA while pDNA from BCTs remained stable. CONCLUSIONS: BCTs prevent increases in gDNA levels that can occur during sample storage and shipping. This new device permits low abundance DNA target detection and allows accurate cfDNA concentrations.

WITHDRAWN: Compatibility of a blood collection tube that stabilizes cell-free DNA with a rapid fluorescence assay.

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Stabilization of cell-free RNA in blood samples using a new collection device.

OBJECTIVE: To investigate whether a new blood collection device stabilizes cell-free RNA (cfRNA) in blood post-phlebotomy when compared to collection using K(3)EDTA tubes. DESIGN AND METHODS: Blood samples were drawn from healthy donors into K(3)EDTA tubes and Cell-Free RNA BCTs (BCTs) and stored at room temperature (20-25 °C). At specified time points (days 0-3), plasma was separated and cfRNA was extracted. Reverse transcription real-time PCR was used to quantify mRNA for c-fos, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and for 18S rRNA. RESULTS: Blood drawn into K(3)EDTA tubes showed a steady increase in RNA concentration over 3 days of ex vivo incubation. Blood drawn into BCTs showed no statistically significant change in RNA copy number except for GAPDH on day 3. CONCLUSIONS: The novel chemical cocktail contained in the new device allows for the stabilization of cfRNA in blood samples at room temperature, which potentially enhances the clinical utility of cfRNA.