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 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. Results Combining 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. 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.

[1]  Joachim Thiery,et al.  Comparison of Whole Blood RNA Preservation Tubes and Novel Generation RNA Extraction Kits for Analysis of mRNA and MiRNA Profiles , 2014, PloS one.

[2]  C. Gieger,et al.  Mapping the Genetic Architecture of Gene Regulation in Whole Blood , 2014, PloS one.

[3]  P. Heath,et al.  Comparison of Blood RNA Extraction Methods Used for Gene Expression Profiling in Amyotrophic Lateral Sclerosis , 2014, PloS one.

[4]  R. Lahesmaa,et al.  Genome-wide comparison of two RNA-stabilizing reagents for transcriptional profiling of peripheral blood. , 2013, Translational research : the journal of laboratory and clinical medicine.

[5]  E. Susser,et al.  Human blood RNA stabilization in samples collected and transported for a large biobank , 2012, BMC Research Notes.

[6]  D. Mehta,et al.  Peripheral blood gene expression: it all boils down to the RNA collection tubes , 2012, BMC Research Notes.

[7]  W. Ahrens,et al.  Influence of sample collection and preanalytical sample processing on the analyses of biological markers in the European multicentre study IDEFICS , 2011, International Journal of Obesity.

[8]  I. Pigeot,et al.  The IDEFICS cohort: design, characteristics and participation in the baseline survey , 2011, International Journal of Obesity.

[9]  R. Rabin,et al.  Systematic method for determining an ideal housekeeping gene for real-time PCR analysis. , 2008, Journal of biomolecular techniques : JBT.

[10]  R. Yeung,et al.  Assessment of sample collection and storage methods for multicenter immunologic research in children. , 2008, Journal of immunological methods.

[11]  K. Raddassi,et al.  Differential gene expression profiles are dependent upon method of peripheral blood collection and RNA isolation , 2008, BMC Genomics.

[12]  Thomas D. Schmittgen,et al.  Analyzing real-time PCR data by the comparative CT method , 2008, Nature Protocols.

[13]  Ajit Varki,et al.  Siglecs and their roles in the immune system , 2007, Nature Reviews Immunology.

[14]  J. Gabert,et al.  Assessment of a new RNA stabilizing reagent (Tempus Blood RNA) for minimal residual disease in onco-hematology using the EAC protocol. , 2006, Leukemia research.

[15]  D. Goldstein,et al.  Reliable gene expression measurements from degraded RNA by quantitative real-time PCR depend on short amplicons and a proper normalization , 2005, Laboratory Investigation.

[16]  N. Rothwell,et al.  Interleukin-1 and neuronal injury , 2005, Nature Reviews Immunology.

[17]  K Dheda,et al.  Real-time RT-PCR normalisation; strategies and considerations , 2005, Genes and Immunity.

[18]  Hui-Rong Qian,et al.  Optimized blood cell profiling method for genomic biomarker discovery using high-density microarray , 2005, Biomarkers : biochemical indicators of exposure, response, and susceptibility to chemicals.

[19]  M. Kubista,et al.  Properties of the reverse transcription reaction in mRNA quantification. , 2004, Clinical chemistry.

[20]  David A Stenger,et al.  Assessment of two methods for handling blood in collection tubes with RNA stabilizing agent for surveillance of gene expression profiles with high density microarrays. , 2003, Journal of immunological methods.

[21]  F. Speleman,et al.  Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes , 2002, Genome Biology.

[22]  M. Pfaffl,et al.  A new mathematical model for relative quantification in real-time RT-PCR. , 2001, Nucleic acids research.