Study design and blood collection
Three healthy male volunteers aged 30-35 years with body mass index of 20-25 were enrolled in the study (see Figure 3). All subjects were in good physical condition and free from medication. Leucocytes counts, C-reactive protein (CRP), gamma glutamyl transpeptidase (GGT), alanine aminotransferase (ALAT) and aspartate aminotransferase (ASAT) levels were not significantly elevated. The optimal time of blood collection for mRNA measurement after dexamethasone stimulation was defined in a pilot experiment. The tmax of orally administered dexamethasone is about 1.5 h with a t1/2 of about 4 h [7]. In our pilot experiment, maximal gene expression was observed 3 and 5 h after dexamethasone intake, hence we used a 3 h interval between blood sampling. Non-stimulated peripheral blood samples were obtained at noon after 2 h of fasting and abstention from coffee, smoking and physical activity, followed by orally administration of 1.5 mg dexamethasone. Stimulated peripheral blood samples were drawn 3 h later. For each sample, duplicate measures of 2.5 ml and 3 ml blood was collected in PAXgene™ tubes (PreAnalytiX GmbH, Hombrechtikon, Switzerland) and Tempus™ tubes (Applied Biosystems, Foster City, CA, USA) respectively. The PAXgene™ and Tempus™ tubes were stored for 2.5 h at room temperature and then transferred to -20°C. Due to the lower RNA yield from the PAXgene™ tubes, RNA from two PAXgene™ tubes was pooled together for the experiment.
Serum, EDTA and Li Herapin tubes (Sarstedt AG, Nümbrecht, Germany) were collected concurrently to analyse differential blood counts, cortisol and ACTH levels and liver enzymes. Administration of 1.5 mg dexamethasone p.o. resulted in a decrease in cortisol and ACTH levels at 3 h post-intake, indicating successful dexamethasone stimulation in all 3 volunteers.
The study was approved by the Ethics Committee of the Ludwig Maximilians University in Munich, Germany, and written informed consent was obtained from all subjects.
RNA extraction, globin reduction and amplification
Total RNA was isolated from whole blood stored in PAXgene™ and Tempus™ tubes according to the respective manufacturer's instructions. The PAXgene™ samples were processed using the PAXgene™ Blood RNA Kit based on the Quiagen method for column purification of nucleic acids (PreAnalytiX GmbH, Hombrechtikon, Switzerland, catalogue number 762174). The Tempus™ samples were isolated using the Tempus™ Spin RNA Isolation Kit (Applied Biosystems, Foster City, CA, USA, catalogue number 4380204). RNA quality was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). The concentration and purity of total RNA was independently assessed by 260/280 UV absorption ratios, respectively (Nanophotometer, Implen, Munich, Germany). RNA samples were divided into non-globin reduced and globin reduced groups. The GLOBINclear™-Human Kit (Ambion, Inc., Texas, USA, catalogue number #AM1980) was used to remove globin mRNA. Sample amplification and labeling for both globin-reduced and non-reduced samples was performed using the IlluminaR TotalPrep RNA Amplification Kit (Ambion, Inc., Texas, USA, catalogue number AMIL1791).
Processing of samples on microarrays
Biotin-labeled cRNA was hybridized to Illumina HumanHT-12 Expression BeadChips (Illumina, Inc., San Diego, CA, USA, catalogue number BD-103-0603) according to manufacturer's protocols. Microarrays were scanned with the Bead Array Reader (Illumina, Inc., San Diego, CA, USA).
qPCR procedures
For validation of the results, cDNA was synthesized from 500 ng total RNA using Superscript II Reverse Transcriptase (Invitrogen, Darmstadt, Germany). qPCR was performed using the Universal Probe Library for TOMM7, RAB37, FKBP5, IL18R1 and TBP on the Roche LightCycler 480 (Roche Applied Science, Penzberg, Germany). Assays were designed using the Probe Finder Software (Roche Applied Science) and run in triplicates in a total reaction volume of 10 μl.
Statistical analysis
Microarray expression analysis was performed in R [16], using beadarray (providing routines to handle Illumina BeadStudio data) [17], limma (for statistical routines) and vsn (for normalization) [18] packages. Raw microarray scan files were exported using the Illumina Beadstudio program and loaded into R for downstream analysis. The data were transformed and normalized using the variance stabilizing normalization method. Of the 48804 probes present on the Human HT-12v3, probes were selected which fulfilled the criteria of Illumina probe detection p-value of < 0.01 in the individuals. Raw qPCR data from the Roche LightCycler 480 System was extracted and the crossing thresholds (CT) from the technical replicates were averaged across all samples. Transcripts were normalized to housekeeping gene TBP.
Significantly regulated genes were detected using generalized linear models and corresponding fold changes between the groups in R. For differential expression analysis, functions from the limma package [19] were used on the normalized log2-transformed expression values before and after dexamethasone challenge; group differences were tested for collection method, globin reduction status, and pre- vs. post dexamethasone stimulation. Results were corrected for multiple testing by 10,000 permutations for each transcript using the permutation of regressor residuals test (PRR) as implemented in the R package glmperm (http://cran.r-project.org/web/packages/glmperm/index.html). Clinical variables and RNA quality measures were compared using a one way ANOVA with SigmaStat® for Windows (Release 2.0, SPSS Inc., Chicago, Illinois 60606, USA).