Dawn Obermoeller, Ambion, Inc.
- From blood to RNA in less than an hour--no need to isolate white blood cells
- RNAlater® included to stabilize RNA
- Samples can be stored for days at ambient temperature
- Recovers highly pure RNA, suitable for demanding applications
Ambion's RiboPure™-Blood procedure overcomes the challenges involved in isolating intact RNA from whole blood. In the past, researchers typically had to isolate white blood cells (WBC) from whole blood prior to RNA isolation to deal with the plethora of contaminants present in blood. One of the challenges involved in isolating RNA from blood is that white blood cells make up only a small fraction (about 0.2%) of blood cells. Red blood cells, which account for the vast majority of cells present in blood, contain large amounts of hemoglobin, which inhibits RT-PCR. Plasma, which comprises ~60% of blood, has a high concentration of RNases, as do some white blood cell subtypes, making it necessary to quickly inactivate RNases in blood samples to preserve the RNA. The large amount of hemoglobin in whole blood further complicates RNA purification by clogging filters, beads and other solid-phase matrices, and if carried over will interfere with downstream applications. The RiboPure-Blood Kit combines two sequential RNA purification techniques (Figure 1) to remove all of these potential contaminants, resulting in high yields of pure RNA directly from whole blood (without prior fractionation of WBCs) that is ideal for RT-PCR (Figure 2), microarrays, or any other application with stringent purity needs.
Figure 1. Schematic of RiboPure™-Blood Procedure. Figure 2. Real-time RT-PCR from RiboPure™-Blood Isolated RNA.
Real-time RT-PCR experiment in which p53, BTK, and GAPDH transcripts were detected in the RNA isolated using the RiboPure-Blood procedure. No genomic DNA was detectable in "no RT" control reactions.
RNA from Whole Blood in Less than One Hour
The simple RiboPure-Blood procedure consists of three steps: 1) lysis with fresh or
-treated whole blood in guanidinium based lysis solution, 2) initial RNA purification by phenol/chloroform extraction, and 3) final RNA purification on a glass fiber filter. The phenol/chloroform extraction step removes proteins from the sample to prevent clogging of the glass fiber filters and eliminate heme contamination. A post-elution DNase treatment can then be performed with the
included in the kit. The entire procedure, including the optional DNase treatment, can be completed in less than 1 hour (Figure 1).
Ideal for Rigorous Applications
By combining the phenol/chloroform extraction, silica matrix purification, and the DNase treatment, the RiboPure-Blood procedure removes protein, heme, genomic DNA, and RNases resulting in high yields of pure RNA that are ideal for RT-PCR or array analysis.
Stabilize RNA in Whole Blood
When collecting blood samples for RNA analysis, storage of the samples prior to RNA isolation may be necessary. The RiboPure-Blood Kit contains RNA
later for the stabilization of RNA in whole blood. The addition of RNA
later to blood samples stabilizes the RNA and prevents its degradation. RNA
later treated samples can be safely stored at ambient temperature for extended periods of time (up to several days). As a result, samples can be safely transported from the point of collection to a distant lab for RNA isolation without RNA degradation.
Everything You Need
The kit provides sufficient reagents for the stabilization and purification of RNA from 40 whole blood samples ranging from 0.3-0.5 ml each. Average yields of RNA are between 2-4 µg/0.5 ml of whole blood (variation is due to donor-specific differences in WBC counts). The RNA purified with the RiboPure-Blood Kit can be used in challenging applications, such as RT-PCR and array analysis.