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

Transcriptome profiling of human whole blood is used to discover biomarkers of diseases and to assess phenotypic traits. Recently, finger-stick blood collection systems have allowed a less invasive and quicker collection of peripheral blood. Such non-invasive sampling of small volumes of blood offers practical advantages. The quality of gene expression data is strictly dependent on the steps used for the sample collection, extraction, preparation and sequencing. Here we have: (i) compared the manual and automated RNA extraction of small volumes of blood using the Tempus Spin RNA isolation kit and the MagMAX for Stabilized Blood RNA Isolation kit , respectively; and (ii) assessed the effect of TURBO DNA Free treatment on the transcriptomic data of RNA isolated from small volumes of blood. We have used the QuantSeq 3' FWD mRNA-Seq Library Prep kit to prepare RNA-seq libraries, which were sequenced on the Illumina NextSeq 500 system. The samples isolated manually displayed a higher variability in the transcriptomic data as compared to the other samples. The TURBO DNA Free treatment affected the RNA samples negatively, decreasing the RNA yield and reducing the quality and reproducibility of the transcriptomic data. We conclude that automated extraction systems should be preferred over manual extraction systems for data consistency, and that the TURBO DNA Free treatment should be avoided when working on RNA samples isolated manually from small volumes of blood.