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

• Isolate leukocytes from whole blood in minutes
• Extract total RNA from leukocytes
• No additional globulin reduction step required
• Stabilize RNA to preserve expression profiles
>• Retains white blood cells for several days at room temperature

The LeukoLOCK™ system is optimized for use with human blood. Blood is a repository of cellular information; however, the presence of globin mRNA in RNA prepared using whole blood can interfere with downstream expression profiling applications. The LeukoLOCK™ system uses filter column-based leukocyte removal technology to separate leukocytes from whole blood and RNAlater™ solutions to stabilize the cells on the filter column. Purification of intrinsic globulin mRNA-depleted RNA from captured leukocytes by excluding red blood cells improves sample utility for expression profiling and other applications (as shown).

Leukocyte capture and stabilization
Pass a 9–10 mL sample of anticoagulated blood through a LeukoLOCK™ filter column, which captures the total leukocyte population while removing red blood cells (including reticulocytes). red blood cells), platelets and plasma. After rinsing with phosphate-buffered saline, the filter column is rinsed with RNAlater™ solution to stabilize RNA in captured leukocytes (see protocol diagram). RNA can be isolated immediately, or stable cells can be left at room temperature for several days or longer at –20°C or –80°C (as shown).

RNA Isolation
RNA is purified by first disrupting the captured cells in a guanidinium thiocyanate-based solution (which releases RNA while inactivating nucleases) . Cell lysates were collected and briefly treated with proteinase K. RNA is then purified from the lysate by washing using MagMAX™ magnetic bead technology, followed by DNase treatment (using TURBO DNase™) and final purification.

Expression Studies
Both quantitative RT-PCR and microarray expression studies have demonstrated the suitability of purified RNA for reliable transcriptome profiling (see figure). RNA isolated using the LeukoLOCK™ System yields longer median aRNA after amplification and a higher percentage of calls than RNA processed using common blood RNA isolation procedures, without the need for additional post-extraction steps to remove globulin mRNA (see figure). Because globin aRNA represents 25–40% of the aRNA peak of total whole blood RNA, understandably lower total RNA yields are obtained when using methods that remove unwanted globin transcripts. RNA amplified after LeukoLOCK™ purification results in greater array sensitivity due to a significant reduction in competing globulin transcripts.

Kit Yield
Recovery rates vary between donors but are typically 10–20 µg RNA/9–10 mL whole blood (as shown in the figure Show). RNA recovered using the LeukoLOCK™ System contains less than 1/10 of the reticulocyte-derived α- and β-globin mRNA found in typical RNA samples from unfractionated whole blood.