High-Throughput Sample Preparation from Whole Blood for Gene Expression Analysis

Blood Collection 

Whole blood (10ml) was collected from two human donors in EDTA-containing Vacutainer tubes (BD). For each donor sample, 300μL aliquots were distributed to 24 wells of a 96-well plate containing 600μL lysis/binding Solution. The blood was stored briefly (<10min) in the collection tubes at room temperature until addition to lysis plate, described below.

RNA Isolation and Analysis 

The MagMAX-96 Blood RNA Isolation Kit protocol was modified to accommodate 300μL whole blood samples, and implemented on the KingFisher 96 Magnetic Particle Processor (Thermo Electron Corp.). The reagent volumes and layout of the KingFisher 96 Processor turntable are listed in Figure 1. The lysis plate was prepared immediately before starting the process to minimize RNA degradation. The KingFisher 96 Processor completed the lysing, washing, genomic DNA digestion, and final RNA purification in <1h.

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Figure 1 MagMAX-96 Blood protocol scaled-up for the KingFisher 96 Processor. The layout of the KingFisher 96 Magnetic Particle Processor turntable is shown with the positions of plates containing appropriate MagMAX-96 Blood Kit reagents (Panel A). Upon completion of a step, the turntable turns clockwise to move the plate containing the next reagent under the magnetic head. The reagent volumes, step times, and plate sizes are also shown (Panel B). The step times include both mixing and bead collection.

RNA yield was quantified by A260 using a UV–Vis spectrophotometer (NanoDrop Technologies), and its integrity was examined by microfluidics capillary electrophoresis with an RNA LabChip Kit on an Agilent 2100 bioanalyzer.

Quantitative RT-PCR (qRT-PCR) targeting PTGS2 and TP53 was performed on a 7900HT Fast Real-Time PCR System using appropriate TaqMan Gene Expression Assays (Applied Biosystems Inc.). An internal control transcript, XenoRNA-01 Control RNA was used to monitor efficiency of qRT-PCR reactions. XenoRNA-01 Control RNA is 1kb long, and shows no sequence homology to any sequence deposited at GenBank.

High-Throughput Globin mRNA Removal 

Globin mRNA removal was performed with 0.3–1μg total RNA samples in triplicate using a GLOBINclear—Human Kit (Ambion Inc.) following the standard protocol from the instruction manual.7 The process was implemented on a Biomek NX Laboratory Automation Workstation (Beckman Coulter) equipped with an MJ Moto Alpha Thermocycler (MJ Research) and a 96-Well Magnetic-Ring Stand (Ambion). The initial 50°C incubation steps were performed in a 96-well PCR plate (Applied Biosystems), and all subsequent steps were performed in a 96-well U-bottom plate. qRT-PCR was used to measure globin mRNA before and after the procedure, normalized to human RNA polymerase II (hRPII) mRNA, to calculate the efficiency of globin mRNA removal.

mRNA Amplification and Biotin Labeling 

Globin-depleted total RNA was amplified and labeled using a MessageAmp II-96 aRNA Amplification Kit (Ambion), which is based on the Eberwine procedure.7 Briefly, total RNA is reverse transcribed using an oligo(dT) primer bearing a T7 promoter sequence. The cDNA undergoes second-strand synthesis and purification to yield a template for in vitro transcription (amplification) with T7 RNA polymerase. Biotin-11-Uridine-52-triphosphate (Biotin-UTP) was used in the transcription reaction to label the aRNA. Magnetic bead-based methods for both cDNA and aRNA purification8 were implemented on a Biomek Liquid Handler (Beckman Coulter). The fully automated procedure has been described elsewhere.9, 10

Microarray Analysis 

Microarray analysis was performed using GeneChip Human Genome U133 Plus 2.0 Arrays (Affymetrix), following the standard protocol recommended by the manufacturer, and expression profiles were analyzed with the manufacturer's standard software.

Total RNA Isolation and Quality Examination 

Endogenous ribonucleases are abundant in whole blood and can quickly degrade RNA, especially upon cell lysis,11 if not effectively eliminated. The MagMAX-96 Blood RNA Isolation Kit uses guanidine thiocyanate to lyse blood cells and to denature nucleases12 while allowing RNA to bind to magnetic beads. Genomic DNA is digested with TURBO DNase under high salt conditions to minimize RNA degradation. For high-throughput preparation of total RNA, magnetic bead-based technology offers the following advantages over glass fiber filter-based methods: (1) the beads can be fully suspended in solution, enabling efficient binding, washing and elution with reduced carryover of inhibitors10, 13; (2) the use of microsized beads permits elution volumes as low as 20μL, resulting in higher final RNA concentrations that are better suited for downstream processes; and (3) it is more reliable for walk-away automation because there is no potential for vacuum failure or clogging seen in filter-based technology.

The MagMAX-96 Blood RNA Isolation Kit is designed for isolation of total RNA from 50μL whole blood in a regular 96-well plate, with a typical yield of ∼0.5μg total RNA. To obtain enough high-quality RNA for microarray analysis, we scaled up the MagMAX-96 protocol to 300μL whole blood. In addition, we implemented processing on a KingFisher 96 Magnetic Particle Processor. As determined by A260, 1.88±0.29μg and 2.06±0.28μg of total RNA were isolated from 300μL blood samples from Donor 1 and Donor 2, respectively (24 replicates each). Variation in RNA yield between donors is common, mainly due to the difference in white blood cell counts among individuals. The consistency of the RNA yield from donor replicates indicates that the RNA isolation process is robust.

To further illustrate the consistency of RNA yields using the scaled-up MagMAX-96 Blood protocol, eight technical replicates of RNA from each donor were randomly selected and qRT-PCR was performed targeting two volatile blood genes (PTGS2 and TP53; Fig. 2). While donor-to-donor variation was significant, as expected, variation among the technical replicates was very low (SD≤0.27Ct) showing that this method is consistent and viable for isolating RNA for gene expression analysis.

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Figure 2 Consistency of RNA isolated using the scaled-up MagMAX-96 Blood protocol. Eight randomly selected technical replicates (5μL each, 10% of the total preparation) of RNA from each donor were used in 15μL qRT-PCR reactions targeting PTGS2 and TP53 with TaqMan primers and probes.

The standard GLOBINclear globin mRNA reduction protocol is suitable for RNA samples up to 14μL. Using MagMAX-96 Blood elution volumes of 50μL, ≥0.5μg total RNA could be used as input for the GLOBINclear protocol without a need for vacuum concentration, allowing the two procedures to be seamlessly linked.

The purity of total RNA isolated from whole blood is a big concern, because whole blood is very rich in proteins, which inhibit reverse transcription.14, 15 To detect the presence of inhibitors in the RNA isolated using the scaled-up MagMAX-96 Blood protocol, we added 0, 2, and 5μL of the extracted RNA to 15μL qRT-PCR control reactions that each contained 1000 molecules of a target XenoRNA-01 Control RNA. As shown in Figure 3, amplification of XenoRNA-01 Control RNA was not inhibited by as much as 5μL extracted RNA (1/3 of the total qRT-PCR reaction volume), demonstrating that inhibitors of qRT-PCR reactions were effectively excluded from the recovered RNA.

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Figure 3 Absence of qRT-PCR inhibitors in RNA isolation using the scaled-up MagMAX-96 Blood protocol. One thousand copies of XenoRNA-01 Control RNA were used in 15μL qRT-PCR reactions targeting XenoRNA-01 Control RNA using TaqMan primers and probes; 0, 2, or 5μL of RNA isolated from six randomly selected samples were added to the reactions.

Because whole blood is rich in nucleases, total RNA isolated from whole blood is often degraded and not suitable for microarray analysis. We examined the integrity of isolated RNA on an Agilent 2100 Bioanalyzer. Twelve samples randomly selected from a 96-well plate had 28S:18S rRNA ratios ≥1.1 (Fig. 4A and B); a ratio of >1.0 is typically considered indicative of RNA with a high degree of integrity for total RNA isolated from whole blood. The RNA Integrity Number (RIN) algorithm analyzes bioanalyzer information from both rRNA bands, as well as information contained outside the rRNA peaks (i.e., potential degradation products), to provide a more complete picture of RNA degradation states. The RIN values for these samples were ≥6.7 (Fig. 4B), generally considered to indicate a high degree of integrity. The A260/A280 ratio for all 12 samples was >1.74 (Fig. 4B), indicating RNA free of contaminating proteins. These measurements suggest that the scaled-up MagMAX-96 Blood protocol delivered RNA sufficiently intact and pure for high-quality microarray analysis.

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Figure 4 Integrity of RNA isolated using the scaled-up MagMAX-96 Blood protocol. Twelve samples of total RNA were selected in a V-shaped pattern from an entire plate of 96 samples processed using the scaled-up MagMAX-96 Blood protocol. Each sample (1.2μL) was analyzed on an Agilent 2100 Bioanalyzer; results are rendered as a gel image (Panel A). The two predominant bands are 28S and 18S rRNA. 28S:18S rRNA ratios and RIN values were calculated using Agilent Technologies 2100 Expert Software (Panel B).

Effectiveness of Globin mRNA Reduction 

Total RNA isolated from whole blood is very rich in α- and β-globin mRNA. A good globin mRNA reduction protocol should effectively remove both globin mRNAs while efficiently recovering the remaining RNA so that the expression profile is maintained. To test these two parameters, three initial quantities of RNA isolated from whole blood were processed in triplicate in the high-throughput GLOBINclear protocol. RNA recovery from the GLOBINclear processing was measured by absorbance at 260nm. As summarized in Table 1, ≥80% of input RNA was recovered in all samples after globin mRNA reduction.

Table 1. Recovery of RNA after globin mRNA reduction

	Input (total RNA, μg)	After globin removal (μg)	% Recovered
	0.98	0.78	79.6
	0.70	0.59	84.3
	0.28	0.27	96.4

Three input quantities of total RNA were processed in triplicate in the high-throughput GLOBINclear—Human protocol. RNA was quantified using a NanoDrop Spectrophotometer.

To quantify the levels of α- and β-globin mRNA, 5μL of both the processed RNA and the original, unprocessed RNA were used in qRT-PCR reactions targeting α-globin, β-globin, and hRPII mRNA. Both α-globin and β-globin amplification showed significant shifts after processing by the GLOBINclear globin reduction method; however hRPII remained unaffected by processing (data not shown). α-Globin and β-globin mRNAs were reduced by >94% and >98%, respectively, with various total RNA input (Table 2). Coefficient of Variance (CV) was ≤6% in all cases, except for β-globin mRNA reduction in the 0.98μg sample (CV=14.5%).

Table 2. Effectiveness of globin mRNA reduction

	Input RNA (μg)	α-Globin		β-Globin
		ΔΔCta Processed/unprocessed	% Remaining	ΔΔCt Processed/unprocessed	% Remaining
	0.98	5.0±0.5	3.3±1.2	7.2±0.5	0.7±0.3
	0.70	4.3±0.1	5.1±0.3	7.1±0.1	0.7±0.1
	0.28	4.2±0.2	5.5±0.7	7.0±0.5	0.8±0.3

Both the processed RNA and the original, unprocessed RNA (5μL each) as described in Table 1 were used in qRT-PCR reactions targeting α-globin, β-globin, and hRPII mRNA. α-Globin and β-globin mRNA levels in processed (depleted) and unprocessed (total) samples were normalized to hRPII.

a ΔΔCt: [Ct(globin, depleted)−Ct(hRPII, depleted)]−[Ct(globin, total)−Ct(hRPII, total)]; Ct: threshold cycle.

The high efficiency of globin mRNA removal, coupled with good RNA recovery, demonstrates that the MagMAX-96 Blood protocol followed by GLOBINclear processing provides an effective method for preparing RNA suitable for successful mRNA amplification, labeling, and expression profiling on a microarray.

mRNA Amplification and Expression Profiling on Microarrays 

The ultimate test of the quality of the isolated RNA and the effectiveness of globin mRNA removal is the quality of the microarray data after mRNA amplification/labeling. The MessageAmp II-96 aRNA Amplification Kit was used to amplify and label RNA on a Biomek Liquid Handler.8, 9, 10 Each aRNA (15μg) was then hybridized to GeneChip Human Genome U133 Plus 2.0 Arrays. On average, samples which had not been processed with the GLOBINclear Kit had 17,052 Present calls, while 19,023 (∼2000 or ∼12% more) genes were called Present in samples which had undergone global mRNA reduction. This is consistent with what is observed when the GLOBINclear protocol is carried out manually in a single tube.16