- Fractionates leukocytes from whole blood in minutes
- Extracts total RNA from leukocytes
- No additional globin reduction steps required
- Stabilizes RNA to preserve expression profile
- Preserves leukocytes at room temperature for days
LeukoLOCK Total RNA Isolation System
The LeukoLOCK Total RNA Isolation System (patent pending) is an innovative method for cellular fractionation of whole blood and total RNA stabilization and extraction from the leukocyte population. The LeukoLOCK System is optimized for use with human blood. Blood is a storehouse of cellular information; however, the presence of globin mRNA in RNA prepared from whole blood can interfere with downstream expression profiling applications. The LeukoLOCK System employs filter-based leukocyte-depletion technology to isolate leukocytes from whole blood and Ambion’s RNAlater® to stabilize the cells on the filter. By excluding red blood cells, the RNA that is purified from captured leukocytes is inherently depleted of globin mRNA, which improves sample utility for expression profiling and other applications (Figure 1).
Leukocyte Capture and Stabilization
A 9–10 ml sample of anticoagulated blood is passed through a LeukoLOCK filter, which captures the total leukocyte population while eliminating red blood cells (including reticulocytes), platelets, and plasma. After rinsing with phosphate-buffered saline, the filter is flushed with RNAlater to stabilize the RNA in the captured leukocytes. (See PROTOCOL SCHEMATIC.) The RNA can be isolated immediately, or stabilized cells can be maintained for several days at room temperature or for longer periods at –20°C or –80°C (Figure 2).
RNA Isolation
Purification of the RNA is done by first disrupting the captured cells in a guanidinium thiocyanate-based solution that releases the RNA while simultaneously inactivating nucleases. The cell lysate is collected and briefly treated with Proteinase K. RNA is then purified from the lysate using Ambion’s MagMAX™ magnetic bead-based technology for washing, followed by DNase treatment (with TURBO DNase™) and final clean-up.
Expression Studies
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 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 Affymetrix 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 RNA 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.
Kit Yields and Contents
Recovery varies from donor to donor, but is typically 10–20 µg of RNA per 9–10 ml of whole blood (Figure 5). RNA recovered using the LeukoLOCK System contains less than 1/10th the amount of reticulocyte-derived alpha- and beta-globin mRNAs present in typical RNA samples from unfractionated whole blood. Thus, the system improved the utility of whole blood RNA for expression profiling and other applications.
The LeukoLOCK System contains reagents to isolate RNA from 20 samples of 9–10 ml of whole blood.
The LeukoLOCK Total RNA Isolation System is also available as two separate modules (see below).
LeukoLOCK Fractionation & Stabilization Kit
This kit contains only the materials and reagents for WBC fractionation and leukocyte stabilization. Stabilized leukocytes can then be stored or shipped to another location. RNA isolation requires purchase of the LeukoLOCK Total RNA Isolation Kit (see below). These kits (modules of the Total RNA Isolation System) are offered separately to accomodate blood sample collection at one location and sample processing at a different location.
LeukoLOCK Total RNA Isolation Kit
This kit contains only the reagents for total RNA isolation from leukocytes captured using the LeukoLOCK Fractionation & Stabilization Kit. These kits (modules of the Total RNA Isolation System) are offered separately to accomodate blood sample collection at one location and sample processing at a different location.
Trademarks, Patents, Licensing
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