Blood is the only human tissue routinely available for RNA expression studies and blood-based biomarker discovery, because it can be collected by relatively non-invasive procedures and is rapidly regenerated. Efficient and robust methods for RNA stabilization and extraction from blood are required for these types of analyses (see sidebar, The Challenges of Analyzing Blood Samples). To address these needs, Applied Biosystems, now including Ambion, has developed several approaches for extracting RNA from blood. Since there is no “one-size-fits-all” solution for blood-based sample preparation, we have developed multiple approaches that allow researchers to choose the system that most closely meets the needs of their specific projects.
Because the RiboPure™-Blood Kit, Tempus™ Blood RNA Tubes, and the LeukoLOCK™ Total RNA Isolation System all have effective RNA stabilization reagents, the two major factors that will influence your choice of systems for sample preparation are blood collection volume and primary downstream application (Figure 1 and below). RNA isolated with any of these systems is suitable for RT-PCR analysis. For microarray analysis, the LeukoLOCK System produces RNA that can be used directly for RNA amplification without the need for subsequent globin mRNA removal. RNA isolated with the RiboPure-Blood Kit or Tempus Tubes can also be used for amplification but requires further processing to reduce the large amount of globin mRNA transcripts expressed in blood (use Ambion GLOBINclear™-Human, Whole Blood Globin Reduction Kit) to maximize percent Present calls. To isolate total RNA that includes RNAs <200 bases (e.g., microRNAs), use a simple protocol modification with the RiboPure-Blood Kit or the LeukoLOCK System.
Figure 1. Major Determinants in Choosing a Sample Preparation Method.
† Use Ambion GLOBINclear™-Human Whole Blood Globin Reduction Kit for the protocol
* See www.ambion.com/techlib/misc/ribo_miRNA.pdf for protocol
§ See www.ambion.com/techlib/misc/leuko_iso.pdf for protocol
RiboPure-Blood Kit (up to 0.5 mL)
The RiboPure-Blood Kit
is designed for use with anticoagulated whole blood (up to 0.5 mL). The sample is mixed with RNAlater® Tissue Collection:RNA Stabilization Solution
in a 2 mL microfuge tube, so it can be stored or shipped over several days at ambient temperature; or it can be kept at –20 or –80ºC for long-term archiving. To extract RNA, the sample is first centrifuged to separate the blood from the RNAlater Solution, which is removed. The sedimented blood is then lysed and extracted using a method that eliminates heme and plasma proteins, which can clog filters and interfere with the use of the RNA as a template for reverse transcription. The RNA is then purified from the aqueous phase by solid-phase extraction onto silica filters. Included in the kit are reagents for post-elution DNase treatment using the TURBO™ DNA-free
reagents, which contain a novel DNase removal resin that avoids the need for heat-inactivation of DNase or time-consuming alcohol precipitation of the RNA. Typical yields of RNA using the RiboPure-Blood Kit are 2–5 µg per 0.5 mL blood. A specialized kit exists for the extraction of RNA from rodent blood (Mouse RiboPure-Blood Kit
), and a modified protocol is available for recovery of small RNAs.
Tempus™ Blood RNA Tubes (3.0 mL)
When blood is drawn into a Tempus Tube, the blood cells are immediately lysed by an RNA Stabilizing Reagent, so that RNases are inactivated and gene expression profiles are "frozen." Gene expression profiles of samples in Tempus Tubes are stabile for five days at room temperature, at least one week at 4ºC, and for at least one year at –20ºC.
LeukoLOCK™ System (3–30 mL)
The LeukoLOCK Total RNA Isolation System provides a novel method for the capture and purification of RNA from the total white blood cell (WBC or leukocyte) population in whole blood. This procedure utilizes patented leukocyte depletion filters that are widely used in blood transfusion therapy. Blood is collected in standard EDTA Vacutainer® tubes, and the suction from an empty evacuated blood collection tube is used to draw the sample through the filter in a closed system. The blood filtrate can be lysed for plasma-based assays such as blood chemistry or viral titers. The filter captures all the WBC subsets, including mature myeloid cells, which would be lost during density gradient centrifugation. The filter is then flushed with RNAlater Solution to stabilize the RNA profile in the intact, captured cells. In contrast, red blood cells (RBC) and reticulocytes pass through the filter, resulting in >90% removal of globin mRNA from the isolated RNA (this level of globin RNA removal is sufficient to rescue low-level gene signals on a microarray without the need for post-extraction globin RNA reduction procedures). The entire process of leukocyte fractionation and stabilization requires <5 minutes and does not necessitate centrifugation, dilution of blood with RBC lysis solution, or any other time-intensive steps that may perturb mRNA profiles. The RNAlater solution-soaked filter devices can be stored at room temperature for several days or at –20ºC for months.
RNA is extracted from the captured cells by lysing cells on the filter and purifying the RNA with Ambion MagMAX™ magnetic beads (included). The kit contains TURBO DNase™ enzyme for the efficient elimination of contaminating genomic DNA. The LeukoLOCK method typically yields 10–20 µg of highly pure total RNA from 9 mL of blood (yields vary by donor). The alternate protocol used for miRNA recovery is based on lysis in TRI Reagent® solution followed by silicon filter purification.