Abstract 6689: Evaluating the influence of blood collection tubes and extraction kits on miRNA recovery in cancer biomarker research

Abstract Cell-free RNAs, especially microRNAs (miRNAs), are emerging as valuable biomarkers in cancer research and other diseases. These small, non-coding RNA molecules circulate in the bloodstream, offering insights into the physiological and pathological states of various organs and tumors. However, the stability of miRNAs in blood samples can be significantly affected by the type of blood collection tube used and the handling of samples. Here, we determine the optimal workflow for miRNA extraction from blood samples by comparing three types of blood collection tubes - citrate, EDTA, and Nucleic Acid BCT (NAC) - paired with three different extraction kits: A, B, and C. The three selected extraction kits differ in their sample lysis mechanisms, which are crucial for minimizing the effects of highly active innate RNases present in plasma. Kit A uses a proprietary lysis reagent containing phenol, allowing for plasma lysis at room temperature and recovering RNA from the aqueous layer following phase separation. Kit B employs two consecutive heated lysis steps at 60 °C with chaotropic agents while Kit C executes plasma lysis at 37 °C using proteinase K and further purifies RNA on an automated magnet-based platform. We observed that when plasma samples were processed within 24 hrs with Kit A per manufacturer’s instructions, both RNA spike-in and endogenous miRNA recoveries were notably compromised regardless of tube type, while the mRNA yield was markedly reduced in samples collected in NAC. We were only able to increase recovery with substantial modifications to Kit A’s protocol. In contrast, when we analyzed plasma samples with Kits B and C per manufacturer’s instructions, we saw consistent isolation of miRNA and mRNA across all three tube types, albeit with slight variations in reproducibility. These data show that the recovery of miRNA and mRNA from plasma samples is significantly influenced by the combination of RNA extraction kit and blood collection tube. The automation capability of Kit C facilitates higher throughput processing, which is advantageous in clinical settings where sample volume and processing time are critical factors. To demonstrate the compatibility of Kit C with NAC, cancer-associated miRNAs, including miR-21-5p and miR-122-5, were quantified in samples collected into citrate, EDTA, and NAC. We observed that these miRNAs are maintained in samples stored in NAC for up to five days, but not those stored in EDTA or citrate. These data underscore the importance of selecting appropriate blood collection tubes and extraction kits to optimize miRNA recovery from plasma samples. The findings indicate that both components significantly influence miRNA quantification. Careful consideration of these factors is crucial for obtaining reliable results in clinical studies involving circulating miRNAs. Nucleic Acid BCT is For Research Use Only. Not for use in diagnostic procedures. Citation Format: Eunju Seong, Nicholas M. George, Jordan LaRue, Jing Li, Lisa Bartron. Evaluating the influence of blood collection tubes and extraction kits on miRNA recovery in cancer biomarker research [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6689.