RiboPure™-Blood RNA Isolation Kit
is optimized for maximum yield of high quality total RNA directly from up to 0.5 ml samples of mouse or rat whole blood (minor procedural modifications are needed for blood volumes ≤ 0.25 ml). The kit combines two methods of RNA purification to remove the contaminants that are common to blood samples (protein, heme, genomic DNA, and RNases) and that can compromise downstream procedures. RNA recovered using the Mouse RiboPure-Blood Kit can be used in the most challenging applications, including real-time RT-PCR and array analysis. White blood cell (WBC) mRNA can subsequently be treated to selectively remove globin transcripts using Ambion’s
Figure 1. High Quality RNA Isolated from Diverse Mouse Strains with the Mouse RiboPure™-Blood Kit. RNA from male and female mice from 9 inbred mouse strains was isolated using Mouse RiboPure-Blood RNA Isolation Kit and analyzed on a denaturing agarose gel stained with ethidum bromide. Each sample represented ~5% of the RNA recovered from a 0.5 ml blood sample, except for the samples marked (*) which were ~0.25 ml. The high quality of the RNA is suggested by inspection of the integrity of the 28S and 18S rRNA bands and the appearance of distinct bands in between these rRNAs.
The Mouse RiboPure-Blood Kit includes microcentrifuge tubes pre-loaded with
® for immediate stabilization of RNA within whole blood samples. Rodent blood can be collected in syringes, capillary tubes, or pipets, and then added directly to the pre-loaded tubes without measuring the sample volume. Treated samples can be safely stored at ambient temperature for several days or at -20°C for long-term storage before RNA isolation.
The RNA isolation method is based on lysis of the blood in a guanidinium-based solution, followed by phenol/chloroform extraction, and final purification using glass fiber filter technology. The instructions provided with the kit also include a slightly modified procedure for recovery of small RNAs, including microRNAs.
Figure 2. Bioanalyzer Profile Showing High Quality Mouse RNA Isolated with the Mouse RiboPure™-Blood Kit.
The Mouse RiboPure-Blood Kit was used to isolate RNA from 0.5 ml blood from a male A/J strain mouse. The aqueous phase recovered after phenol/chloroform extraction was split into two aliquots. One was processed for recovery of total RNA, including miRNA and other small RNAs, and the other for recovery of total RNA which is depleted of small RNAs. An aliquot from each preparation separated on an Agilent® 2100 bioanalyzer shows the specificity of each protocol. RNA Integrity Number (RIN) and rRNA ratios, indicators of RNA integrity, are shown on the right. The peak of low molecular weight RNA that corresponds to small RNAs, including microRNAs is indicated.
RNA can be isolated from whole blood in ~35 min using the Mouse RiboPure-Blood Kit. Typicall 30–50 µg of total RNA (including miRNA) is obtained from 0.5 ml mouse blood. (RNA yields from mouse blood are considerably higher than from human blood.) Yields of RNA from rat blood are approximately 18–40 µg from 0.5 ml blood.
Juanita Gonzales, Jennifer Ho, Marianna Goldrick • Ambion, Inc.