Blood collection and RNA isolation
Unfractionated whole blood collection and RNA isolation were performed using the PAXgene Blood RNA Isolation System (PreAnalytiX, a Qiagen BD Company, Valencia, CA, USA). PaxGene tubes contain a proprietary reagent to reduce intracellular RNA degradation and minimize induction of gene expression . With the approval of the OHSU Institutional Review Board, blood was collected from four healthy donors using eight PAXGene tubes per individual. After incubation for two hours at room temperature, four PAXGene tubes per donor were transferred to a -80°C freezer. RNA was isolated from the remaining tubes on the day of collection and from the frozen tubes after storage at -80°C for at least one week. DNase-treatment was performed as per PAXGene manufacturer's recommendation. After pooling samples from 4 frozen or 4 fresh PAXGene tubes for each donor, RNA recovery and quality were assessed by examining UV 260/280 absorbance ratios and RNA size distribution on RNA Nano LabChips (Agilent Technologies, Santa Clara, CA, USA) processed on the Agilent 2100 Bioanalyzer. An RNA Integrity Number (RIN) was generated for each Bioanalyzer trace using Expert software (Agilent). For inclusion in this study, a minimum of 12 μg of RNA per donor from both fresh and frozen blood preparations were required in order to have sufficient material for RNA quality assessments and manufacturer recommended inputs for each labeling protocol. Samples from three of the four donors met the quantitative and qualitative criteria.
Target preparation methods overview
Amplifications and labeling were performed for each donor using one of the following four methods with RNA extracted from fresh and frozen blood samples: Method 1 - Affymetrix one-cycle cDNA synthesis/Affymetrix in vitro-transcription (IVT) on whole blood RNA (no depletion_Affymetrix); Method 2 - Affymetrix one-cycle cDNA/Affymetrix IVT with globin peptide nucleic acids (PNAs) inclusion during cDNA synthesis (Globin PNAs_Affymetrix); and Method 3-RNA pre-treated with Ambion GLOBINclear beads to reduce globin transcripts prior to labeling with the Affymetrix one-cycle cDNA synthesis/Affymetrix IVT (GLOBINclear_ Affymetrix). In the fourth method tested, cDNA target was synthesized from whole blood RNA using the NuGEN Ovation Biotin RNA Amplification and Labeling System v1 (no depletion_NuGEN). Total RNA input amounts were based on manufacturer's recommendations for each method.
Method 1: Affymetrix one-cycle cDNA synthesis/Affymetrix IVT (no depletion_Affymetrix)
Method 1 employed the standard laboratory method for amplification and labeling of total RNA. Target synthesis was performed following the Affymetrix GeneChip Expression Analysis Technical Manual, rev. 5 http://www.affymetrix.com/support/technical/manual/expression_manual.affx with minor modification. Using 1 μg of total RNA as input, messenger RNA was amplified and labeled in two steps. In the first step, mRNA was converted to double-stranded cDNA and purified by phenol-chloroform-isoamyl alcohol extraction and ethanol precipitation. In the second step, amplified and biotinylated cRNA (the target) was produced by in vitro transcription (IVT). Unincorporated nucleotides were removed using the RNeasy Mini kit (Qiagen) followed by an ethanol precipitation of the labeled target.
Method 2: Affymetrix one-cycle cDNA synthesis/Affymetrix IVT with PNA treatment of total RNA (Globin PNAs_Affymetrix)
The standard Affymetrix amplification and labeling was performed with the following modification: four peptide nucleic acid (PNA) oligonucleotides (Applied Biosystems, Foster City, CA, USA) designed to anneal to human α- and β-globin mRNA were added to the total RNA preparations immediately prior to initiation of the target synthesis step in order to bind globin mRNA and block cDNA synthesis. The PNA oligonucleotide stocks were prepared on day of use at concentrations recommended by Affymetrix http://www.affymetrix.com/support/technical/byproduct.affx?product=bloodrna. Globin reduction PNA master mix was prepared by adding equal amounts of the globin PNA stocks. Five μg of total RNA were incubated with oligo-dT primer and 1 μl PNA master mix for 10 minutes at 70°C prior to first strand cDNA synthesis; all subsequent steps for target amplification and labeling were as described for Method 1.
Method 3: Affymetrix one-cycle cDNA synthesis/Affymetrix IVT with GLOBINclear treated total RNA (GLOBINclear_Affymetrix)
Globin mRNA depletion was performed in three steps using the GLOBINclear Kit (Ambion, Austin, TX, USA). First, species-specific biotinylated oligonucleotides complementary to human α- and β-globin mRNA were hybridized with total RNA (5 μg). Second, magnetic streptavidin-coated beads were added to bind the biotinylated-oligonucleotide:globin RNA complexes. Finally, a magnet was used to remove beads with the selectively bound α- and β-globin transcripts. After globin mRNA depletion, RNA quantity was determined by UV absorbance and RNA integrity was assessed using RNA Nano LabChips as described above. RNA recovery following GLOBINclear treatment ranged from 2–5 μg. Target labeling and amplification steps were then performed as described for Method 1.
Method 4: NuGEN Ovation Biotin RNA Amplification and Labeling System v.1 (no depletion_NuGEN)
Ovation v1 labeling and amplification reagents were obtained from NuGEN Technologies, Inc (San Carlos, CA, USA) and biotinylated cDNA targets were prepared according to manufacturer's instructions. Double-stranded cDNA was synthesized from 30 ng total RNA, followed by a linear isothermal amplification (SPIA Amplification™) to produce single-stranded cDNA (target). A proprietary fragmentation and direct labeling process attached biotin to the amplified target. Target purifications were performed using DNA Clean and Concentrator - 25 (Zymo Research, Orange, CA, USA) and the DyeEx 2.0 Spin Kit (Qiagen).
Array hybridization and processing
Targets labeled with Affymetrix one-cycle cDNA synthesis/Affymetrix IVT (Methods 1, 2 and 3) were chemically fragmented as per Affymetrix's recommendations and combined with Affymetrix biotinylated hybridization controls (oligomer B2 and cRNAs for BioB, BioC, BioD and CreX) in hybridization buffer. Hybridizations were performed for 16 hours at 45°C after addition of 6.5 μg of target to the GeneChip Human Genome Focus array (Affymetrix), containing 8700 probe sets. Post-hybridization array processing was performed according to manufacturer's recommendations. The distribution of fluorescent material on the processed array was determined using the Affymetrix 3000 GeneArray laser scanner with the 7G upgrade. Image inspection was performed manually immediately following each scan.
For targets labeled with NuGEN Ovation system (Method 4), the hybridization and processing outlined above was performed with the following modification: following NuGEN's recommendations, 1.3 μg of cDNA target were mixed with Affymetrix hybridization controls in hybridization buffer and hybridized with the Human Genome Focus array for 18 hours at 45°C.
Microarray data quality assessment
The array image scan was processed with Affymetrix Microarray Suite, version 5.0, (MAS 5.0) software. GeneChip expression Arrays contain control probe sets for both spiked and endogenous RNA transcripts (e.g., BioB, BioC, BioD, CreX and species-specific actin and GAPDH). Following absolute analysis of the array pattern with MAS, six values were examined to assess overall assay performance: background, noise, average Signal, % Present, ratio of Signal values for probe sets representing the 5' and 3' ends of actin and GAPDH transcripts, and total Signal for probe sets for BioB, BioC, BioD and CreX. Assays demonstrating poor or marginal performance were flagged.
Quantitative RT-PCR was performed on RNA from donors C and D. cDNA was prepared from selected RNA samples (1 μg each) with a RT2 PCR Array First Strand Kit (SuperArray Bioscience, Frederick, MD, USA) following the manufacturer's instructions. The product was combined with SuperArray PCR SYBR Green master mix and pipetted into all wells of a 96-well RT2Profiler™ PCR Array for the human NF-κB pathway (SuperArray APHS-025A) per the manufacturer's instructions. After 10 min at 95°C to activate the HotStart DNA polymerase, the samples were subjected to 40 cycles of 15 seconds 95°C, 35 seconds at 55°C during which time the SYBR Green fluorescence was recorded, and 30 seconds at 72°C with a Chromo4 thermocycler (BioRad Laboratories, Hercules, CA, USA). Cycle threshold (Ct) values were obtained for each sample and were normalized for each plate by subtracting the mean Ct value for five housekeeping genes (β2-microglobin, hypoxanthine phosphoribosyltransferase 1, ribosomal protein L13a, glyceraldehyde-3-phosphate dehydrogenase, and β-actin) to yield ΔCt values.
For comparison of RT-PCR results with the microarray data, 75 genes from the SuperArray NF-κB pathway panel were matched to probe sets on the Affymetrix Human Genome Focus array.
Data analysis and visualization
Low-level analysis (background correction, normalization, and gene summarization) of microarray data was performed with both Microarray Suite 5.0 (MAS 5.0) [17, 18] and Robust Multi-Array Average (RMA) [19, 20]. Individual arrays were analyzed and scaled with MAS 5.0 using manufacturer's default thresholds for detection calls. For RMA analysis, arrays were normalized together and in groups based on target labeling method using RMA implemented in Affymetrix Expression Console. All microarray data have been deposited in Gene Expression Omnibus (GEO) under accession # GSE13292.
Exploratory data analysis was performed on probe cell intensities and summarized probe set signal values. Box plots of signal distributions were generated with Expression Console (Affymetrix). Scatter plots and correlation measurements were performed using Statistica 6.0 (StatSoft, Tulsa, OK, USA). Venn diagrams were generated using VENNY, an interactive tool for comparing lists with Venn diagrams