Abstract
AimsPrior 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.MethodsThe established PAXgeneTM blood collection method (Qiagen) was compared with the more recent TempusTM 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.ResultsCombining different sampling methods and extraction kits caused strong variations in gene expression. The use of PAXgeneTM and TempusTM 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 PAXgeneTM RNA extraction system from samples collected by TempusTM collection tubes. Comparing only the Norgen RNA extraction methods, RNA from blood collected either by the TempusTM or PAXgeneTM collection system delivered sufficient amount and quality of RNA, but the TempusTM collection delivered higher RNA concentration compared to the PAXTM collection system. The established Pre-analytix PAXgeneTM RNA extraction system together with the PAXgeneTM blood collection system showed lowest CT-values, i.e. highest RNA concentration of good quality. Expression levels of all tested genes were stable and reproducible.ConclusionsThis 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.
Highlights
The use of PAXgeneTM and TempusTM collection systems resulted in RNA of good quality and quantity for the respective RNA isolation system
It was not possible to extract sufficient RNA of good quality with the PAXgeneTM RNA extraction system from samples collected by TempusTM collection tubes
Comparing only the Norgen RNA extraction methods, RNA from blood collected either by the TempusTM or PAXgeneTM collection system delivered sufficient amount and quality of RNA, but the TempusTM collection delivered higher RNA concentration compared to the PAXTM collection system
Summary
A reliable analysis of gene expression is still challenging due to the various available blood sampling as well as subsequent RNA isolation methods, which result in robust changes of expression patterns. Gene expression analysis in peripheral blood is an important and obligatory tool in molecular research and diagnostics, especially in large epidemiological studies. Two major commercially available blood collection tube systems were available and compared in this study, (1) the PAXgeneTM and (2) the TempusTM collection tubes. The PAXgeneTM collection tubes (Preanalytix, Hombrechtikon, Switzerland) are loaded with a patented blend solution, which protects RNA molecules from degradation by RNases and prevents the induction of further gene expression. The TempusTM collection tubes (Life Technologies, Darmstadt, Germany) are preloaded with guanidine and detergent, which stabilizes the RNA by lysing the blood cells and inactivating the RNAses through chaotropic properties
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