Influence of storage conditions of small volumes of blood on immune transcriptomic profiles

Abstract

Objective: Transcriptome analysis of human whole blood is used to discover biomarkers of diseases and to assess phenotypic traits. Here we have collected small volumes of blood in Tempus solution and tested whether different storage conditions have an impact on transcriptomic profiling. Fifty µl of blood were collected in 100µl of Tempus solutions, freezed at − 20 °C for 1 day and eventually thawed, stored and processed under five different conditions: (i) − 20 °C for 1 week; (ii) +4 °C for 1 week; (iii) room temperature for 1 week; (iv) room temperature for 1 day, − 20 °C for 1 day, room temperature until testing at day 7, (v) − 20 °C for 1 week, RNA was isolated and stored in GenTegra solution. We used 272 immune transcript specific assays to test the expression profiling using qPCR based Fluidigm BioMark HD dynamic array. Results: RNA yield ranged between 0.17 and 1.39µg. Except for one sample, RIN values were > 7. Using Principal Component Analysis, we saw that the storage conditions did not drive sample distribution. The condition that showed larger variability was the RT-FR-RT (room temperature-freezing-room temperature), suggesting that freezing-thawing cycles may have a worse effect on data reproducibility than keeping the samples at room temperature.

Technical assessment of different extraction methods and transcriptome profiling of RNA isolated from small volumes of blood

“…Nevertheless, technical improvements are required to make the gene expression profiling of small volumes of blood a reliable and reproducible technique 12 . Automated workflows offer several advantages for large-scale projects, as they increase sample throughput and reduce cost and manual errors 13 – 16 . The MagMAX for Stabilized Blood RNA Isolation kit (Thermo Fisher Scientific, MA, USA) employs a magnetic bead-based technology to purify RNA from blood stored in Tempus solution.…”