Evaluation of Existing Methods for Human Blood mRNA Isolation and Analysis for Large Studies

AIMS: Prior to implementing gene expression analyses from blood to a larger cohort study, an evaluation to set up a reliable and reproducible method is mandatory but challenging due to the specific characteristics of the samples as well as their collection methods. In this pilot study we optimized a combination of blood sampling and RNA isolation methods and present reproducible gene expression results from human blood samples. METHODS: The established PAXgene(TM) blood collection method (Qiagen) was compared with the more recent Tempus(TM) collection and storing system. RNA from blood samples collected by both systems was extracted on columns with the corresponding Norgen and PAX RNA extraction Kits. RNA quantity and quality was compared photometrically, with Ribogreen and by Real-Time PCR analyses of various reference genes (PPIA, β-ACTIN and TUBULIN) and exemplary of SIGLEC-7. RESULTS: Combining different sampling methods and extraction kits caused strong variations in gene expression. The use of PAXgene(TM) and Tempus(TM) collection systems resulted in RNA of good quality and quantity for the respective RNA isolation system. No large inter-donor variations could be detected for both systems. However, it was not possible to extract sufficient RNA of good quality with the PAXgene(TM) RNA extraction system from samples collected by Tempus(TM) collection tubes. Comparing only the Norgen RNA extraction methods, RNA from blood collected either by the Tempus(TM) or PAXgene(TM) collection system delivered sufficient amount and quality of RNA, but the Tempus(TM) collection delivered higher RNA concentration compared to the PAX(TM) collection system. The established Pre-analytix PAXgene(TM) RNA extraction system together with the PAXgene(TM) blood collection system showed lowest C(T)-values, i.e. highest RNA concentration of good quality. Expression levels of all tested genes were stable and reproducible. CONCLUSIONS: This study confirms that it is not possible to mix or change sampling or extraction strategies during the same study because of large variations of RNA yield and expression levels.