Ambion’s new LeukoLOCK™ Total RNA Isolation System (patent pending) provides a novel method for the capture and purification of intact RNA from the white blood cell (leukocyte) population in whole blood (Figure 1). This procedure uses leukocyte depletion filters, which have been widely used in blood transfusion therapy to remove donor leukocytes and prevent graft-versus-host disease in the recipient. The LeukoLOCK System includes disposable leukocyte depletion filters designed to capture total leukocytes from 9-10 ml blood samples; however, the sample volume (range, 3–30 ml) is flexible.
Figure 1. High RNA Quality and Yield Obtained Using the LeukoLOCK™ System
. RNA was purified from a 9 ml blood sample from a healthy male donor using the LeukoLOCK System on the day of blood collection. Total RNA yield was measured by a NanoDrop® Spectrophotometer, and RNA quality (A–B) was determined by analyzing RNA (100 ng) with Agilent® 2100 bioanalyzer expert software.
Blood is collected in standard EDTA Vacutainer® Tubes. A vented transfer spike is inserted into this tube and attached to the filter device (See sidebar, Simple Leukocyte Filtration from Whole Blood). The assembly is then connected to an empty 10 ml evacuated blood collection tube, which provides the suction needed to draw the blood through the filter without opening the tube. A 9–10 ml blood sample can be filtered in about 2 minutes. The filter is then flushed with PBS to eliminate residual red blood cells and with
to stabilize the RNA in the intact, captured cells. The entire process of leukocyte fractionation and stabilization requires less than 5 minutes and is free from centrifugation and other time-intensive steps that may perturb mRNA expression profiles. The RNAlater-treated filters can be stored at room temperature for several days or at -20°C for months with no loss in RNA integrity (Figures 2–3).
Figure 2. RNA from White Blood Cells Can Be Archived on LeukoLOCK™ Filters for At Least a Month.
Total RNA was isolated from replicate whole blood samples (9 ml) from 2 donors. Samples were either processed immediately using the LeukoLOCK protocol, or leukocytes were trapped and stored on filters soaked with RNAlater® for 32 days at –20°C before RNA purification.
Average RNA yields and quality from the two donors were determined using NanoDrop® Spectrophotometer and Agilent® bioanalyzer expert software.
Total RNA was analyzed in quantitative, one-step qRT-PCR (10 µl reactions) using MessageSensor™ RT with 4 different TaqMan® Gene Expression Assays (Applied Biosystems) (input RNA amounts: 50 pg for 18S rRNA, and 50 ng for Casp-1, IFNG, and IL-8). Assays were performed on a Stratagene® Mx3000P™ (standard cycling conditions). Ct values are represented as averaged triplicates. No genomic DNA was detectable in “no RT” control reactions (data not shown). Three of the above transcripts are known to be labile in ex vivo blood : Casp-1=Caspase-1, IFNG=Interferon-gamma, IL-8=Interleukin-8.
Figure 3. Global Expression Profile is Stable at Ambient Temperatures After White Blood Cells were Stored for At Least Three Days on LeukoLOCK™ Filters.
RNA was prepared using the LeukoLOCK Total RNA Isolation System from filtered leukocytes processed immediately or stored on the LeukoLOCK Filter in the presence of RNAlater® for 3 days at room temperature.
RNA yields and quality were determined by a NanoDrop® Spectrophotometer and Agilent® 2100 bioanalyzer expert software.
Total RNA (1 µg, no globin reduction treatment) was amplified using Ambion’s MessageAmp™ II-96 aRNA Amplification Kit. Fragmented aRNA was then hybridized to Human Focus Arrays (Affymetrix®) and scanned with a GeneChip® Scanner 3000. Data were captured and analyzed on GeneChip Operating Software (Affymetrix). A correlation plot from the normalized data is shown for the average array signal intensities for day 0 vs. day 3 biological replicates. (C) Percent Present calls, and GAPDH and b-actin 3' to 5' ratios were also assessed.
RNA is extracted from the captured cells by flushing the filter with a guanidinium-based lysis solution and purifying the RNA with Ambion’s MagMAX™ magnetic bead-based RNA purification technology. The kit includes
for efficient elimination of genomic DNA. The LeukoLOCK method typically yields 10–20 µg of highly pure total RNA from 9 ml of blood (yields will vary among donors). Since the capture filter is selective for leukocytes, red blood cells including reticulocytes pass through, resulting in >90% reduction of globin mRNA in the isolated RNA. This level of globin RNA removal is sufficient to rescue low mRNA expression signals on a microarray and avoids the need for a globin mRNA reduction procedure after RNA extraction.
Both quantitative RT-PCR and microarray expression studies have validated the suitability of the purified RNA for reliable transcriptome profiling. For example, for samples processed immediately or stored frozen for 32 days in LeukoLOCK filters, Ct values were the same in quantitative RT-PCR experiments for four different gene targets, including transcripts known to be extremely labile in ex vivo blood (Figure 2) . Excellent reproducibility was also obtained in microarray experiments for blood samples that were processed immediately or stored for 3 days at room temperature (Figure 3). Compared to RNA processed with a commonly used blood RNA isolation procedure, RNA isolated with the LeukoLOCK System produces longer median aRNA after amplification and higher percent Present calls (by ~8 %, representing >700 additional genes on Affymetix® Human Focus Arrays) without requiring additional post-extraction steps to remove globin mRNA (Figure 4). Since globin aRNA represents 25–40% of the aRNA peak from whole blood total RNA, the yield of total aRNA is understandably lower with methods that deplete unwanted globin transcripts. RNA amplified after LeukoLOCK purification offers greater array sensitivity due to the dramatic reduction in competing globin transcripts.
Figure 4. Microarray Data Comparing LeukoLOCK™ Protocol and a Market Leader Protocol.
Duplicate total RNA samples were purified from pooled, human blood by two RNA isolation methods (biological replicates using LeukoLOCK or Market Leader's System). (A) Samples (1 µg) were directly amplified without globin reduction and labeled with MessageAmp™ II-96 and then analyzed on Affymetrix® Human Focus Arrays. (B–C) The normalized data for each replicate are plotted against the other to create the signal concordance graph. The LeukoLOCK System produced longer median aRNA. Use of this aRNA on the Human Focus Arrays resulted in an 8% increase in percent Present calls.
Juanita Gonzales, Jon Kemppainen, Gary Latham, Marianna Goldrick • Ambion, Inc.