Comparative stability analysis of RNA in dried blood spots for real-time PCR based molecular testing

Dried blood spot (DBS) sampling represents a minimally invasive, cost-effective alternative to venous blood collection, and advantageous for resource-limited diagnostic settings. The reliability of RNA-based molecular assays using DBS depends on RNA stability under various storage conditions. This study evaluated RNA stability in DBS samples stored at varying temperatures and time points, with and without RNAlater pretreatment of Whatman 903™ protein saver cards to determine their suitability for quantitative PCR (qPCR)-based applications. Whole blood was applied onto standard and RNAlater-pretreated (10 μL; 10 spots/card) Whatman 903™ cards and stored at room temperature (RT), 4 °C, and - 20 °C for 1, 3, and 7 days. Total RNA was extracted, quantified, and assessed for purity prior to cDNA synthesis. ABL1 gene amplification was performed using SYBR green real-time PCR. Analytical agreement between matrices for RNA quantity was assessed using Bland-Altman plot, and cycle threshold (Ct) values were compared. RNA yield and purity varied across storage conditions. The highest RNA yield was observed in RNAlater-pretreated DBS cards stored at RT for 7 days (36.3 ± 0.665 ng/μL), with acceptable purity (A260/280 range: 1.7-2.0). On day 7, whole blood samples stored at 4 °C demonstrated reduced RNA yield compared to treated and untreated DBS cards irrespective of the storage temperature. Consistent ABL1 amplification confirmed preserved RNA integrity in both untreated and treated DBS samples (CV <1%), with improved stability in pretreated cards compared to whole blood (CV < 3%). RNAlater pretreatment enhances RNA stability in DBS, supporting its application for reliable qPCR-based molecular diagnostics in routine and field laboratory settings, especially when concordance is established between venous and capillary sampling.