RNA Extraction from Blood: Choosing the Best System for Your Gene Expression Studies

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.

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.

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.