LeukoLOCK™ Total RNA Isolation System

Catalog number: AM1923

Ambion™  Related applications: RNA Extraction

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Description

The LeukoLOCK™ Total RNA Isolation System is an innovative method for cellular fractionation of whole blood and total RNA stabilization and extraction from the leukocyte population. The kit is comprised of a LeukoLOCK™ Fractionation & Stabilization Kit (also available separately; Cat. No. AM1933) and a LeukoLOCK™ Total RNA Isolation Kit.

• 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

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 RNAlater® solution 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 (see Figure).

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® solution 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 (see Figure).

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 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 (see Figure). 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 without requiring additional post-extraction steps to remove globin mRNA (see Figure). 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
Recovery varies from donor to donor, but is typically 10–20 µg of RNA per 9–10 mL of whole blood (see Figure). 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.
For Research Use Only. Not for use in diagnostic procedures.

Specifications

Sample Type (General): Blood, Cells
High Throughput Compatibility: Not High Throughput-Compatible (Manual)
Downstream Application: Reverse Transcriptase PCR (RT-PCR), Northern Blotting, Real-Time Quantitative PCR (qPCR), Microarray Analysis, Nuclease Protection Assays, cDNA Library Construction
Product Size: 20 preps
Final Product: Total RNA
Isolation Technology: Magnetic Bead, Leukocyte Capture Filter
Starting Material (Amount): Up to 10 ml whole blood

Contents & storage

Store 1X PBS pH 7.4, RNAlater;® Solution, Rubber Septum Cap, White Slip Connector, Transfer Spike, LeukoLOCK™ Filter, Elution Solution, LeukoLOCK™Lysis⁄Binding Solution, LeukoLOCK™pH Adjustment Solution, Processing Tubes, Wash Solution 1 Concentrate, Wash Solution 2⁄3 Concentrate and Nuclease–Free Water at room temperature. Store RNA Binding Beads at 4°C. Store 1X LeukoLOCK™DNase Buffer, Proteinase K and TURBO™DNase at -20°C.

Documents

Manuals & protocols

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