Optimizing RNA Extraction from EDTA Blood

For RNA extraction from original Paxgene tubes, samples were thawed on ice for at least 2 h and PAXgene Blood RNA Kit (PreAnalytix, Hombrechtikon, Switzerland) was used to extract blood RNA according to the manufacturer’s manual. This method is referred to as Paxgene Control or PP. For EDTA tubes, we had two strategies to optimise the RNA quantity and quality. The first was to replicate and optimise a reference protocol based on transferring EDTA blood to PAXgene tubes before extraction. Several conditions were then tested including (1) Different extraction kits: PAXgene Blood RNA Kit (PreAnalytix, Hombrechtikon, Switzerland) versus MagMAX blood RNA Isolation Kit for PAXgene (Thermofisher Scientific, MA, USA), (2) different post-transfer incubation time: 2 h versus overnight, and (3) same day extraction vs refreezing the samples for later extraction. The second strategy was to optimise the Nucleospin blood RNA extraction kit (Macherey–Nagel, Westfalen, Germany), which was primarily meant to be used for fresh blood but has been shown to produce a much higher yield of RNA compared to other protocols using frozen blood^{15 (link),16 (link)}.