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Extracting Total RNA from the Blood of Aquatic Species
EcoArray, Inc. is a genomics company that is bringing the revolutionary technology of microarrays to the field of environmental toxicology. Their collaborators focus on almost every species imaginable, from fish to mammals, to evaluate the effects of environmental or research compounds. Since many animals used to evaluate chemical effects are too valuable to sacrifice for tissues, blood samples are taken. Ambion’s RiboPure™-Blood Kit works perfectly for mammals with non-nucleated red blood cells. Recently, scientists at EcoArray have optimized this kit for use with non-mammalian vertebrate species that have nucleated red blood cells (e.g., aquatic animals).
Barbara J Carter, April M Green, and Patrick M Larkin
EcoArray, Inc.
12085 Research Drive • Alachua, FL 32615
Phone: 386-418-1400 • Fax: 386-462-0875
Blood Collection
Whole blood (1–10 ml) from different species (mammals: dolphin and dog; non-mammalian vertebrates: largemouth bass and bonnet head shark) was collected in tubes containing potassium EDTA anticoagulant (“purple-tops”) and inverted several times to mix. The blood was immediately transferred to a sterile tube (5, 15, or 50 ml tube) containing RNAlater® stabilizing solution at a minimum ratio of
0.5 ml blood per 1.3 ml RNAlater (~28% blood). The stabilized blood was placed on ice or put into the freezer
(-20°C) until extraction.
RNA Processing
The RiboPure-Blood protocol consists of three easy steps. First, the sample is centrifuged to pellet the blood cells, the supernatant is removed, and Lysis Solution is added to the cell pellet. Second, there is an initial purification of the RNA by acid-phenol:chloroform extraction. Third, the sample is passed through a glass fiber filter (spin column) for final purification of total RNA. See sidebar, RNA Isolation from Aquatic Species, for protocol adaptations for processing samples with nucleated red blood cells.
A 500 µl aliquot of stabilized whole blood (blood plus RNAlater) was processed for each species. The RiboPure-Blood protocol was followed as written, except two extractions with acid-phenol:chloroform were performed because several of the samples, especially shark, have a very high lipid content. Samples were passed through the filter cartridge and washed as usual. Samples were eluted twice with 50 µl preheated Elution Solution (total volume = 100 µl), and DNase treated as per protocol (nucleated samples will contain RNA and genomic DNA).
Results: High Purity and Yield
Figures 1 & 2 show the purity and concentration of the samples, as well as the calculated yield from 0.5 ml of whole blood. The RiboPure-Blood Kit makes it easy to consistently isolate high-quality RNA required for microarray studies. Samples that are drawn and immediately transferred to RNAlater show A260/A280 ratios of ~1.8-2.0.
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Figure 1. Results of Extracting 500 µl Aliquot of Whole Blood in RNAlater®. Concentration and purity were determined for the undiluted samples using an Eppendorf® BioPhotometer. Based on the initial volumes of blood drawn and RNAlater in the tube, the yield for 0.5 ml of whole blood was calculated. |
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Figure 2. Total RNA Quality from Multiple Species Using the RiboPure™-Blood Kit. 500 ng of DNase-treated total RNA was mixed with one-half volume of formaldehyde loading dye, heated for 10 mins at 70ºC, and run on an ethidium bromide stained denaturing agarose gel. Notice that the 28S rRNA band varies in size. Lane A = Bonnet head shark, Lane B = Largemouth bass, Lane C = Largemouth bass, Lane D = Dolphin, Lane E = Poodle. |
The RiboPure-Blood Instruction Manual states that “Yields of total RNA typically range from ~2–4 µg from 0.5 ml whole blood from normal healthy donors.” This held true for the terrestrial mammalian sample (i.e., poodle: yield of 5.4 µg). In the non-mammalian species, shark and largemouth bass, the red blood cells are nucleated, and the amount of RNA obtained from these blood samples was four to five times higher than that of terrestrial mammalian blood. More importantly, the dolphin sample was unique, showing a high yield similar to shark. These results are consistent with those found in the past for dolphins. Previously, EcoArray scientists extracted RNA from five additional dolphin samples within days of the blood collection and stabilization steps. The yields were similar: 16–20 µg total RNA per 0.5 ml blood (data not shown). Other research on dolphins suggests that the blood of these animals, and marine mammals in general, is unique in other ways.
Acknowledgements
EcoArray, Inc. would like to thank the following people for contributing blood samples:
•Dr. Gregory D Bossart, Harbor Branch Oceanographic Institution, Ft. Pierce, FL
•Dr. Jim Gelsleichter, Mote Marine Laboratory, Sarasota, FL
•Dr. Jeffery A Philpot, Newberry Animal Hospital, Newberry, FL
•Kevin Roberts, Marineland of Florida, St. Augustine, FL
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