Acetone preservation: a practical technique for molecular analysis.
In attempts to establish a convenient and reliable method for field collection and archival preservation of insects and their endosymbiotic microorganisms for molecular analysis, acetone, ethanol, and other organic solvents were tested for DNA preservability of the pea aphid Acyrthosiphon pisum and its intracellular symbiotic bacterium Buchnera sp. After 6 months' storage, not only the band of high-molecular-size DNA but also the bands of rRNA were well preserved in acetone, ethanol, 2-propanol, diethyl ether and ethyl acetate. Polymerase chain reaction (PCR) assays confirmed that the DNA of both the insects and their symbionts was well preserved in these solvents. In contrast, methanol and chloroform showed poor DNA preservability. When water-containing series of acetone and ethanol were examined for DNA preservability, acetone was apparently more robust against water contamination than ethanol. Considering that most biological materials contain high amounts of water, acetone may be a more recommendable preservative for DNA analysis than ethanol which has been widely used for this purpose. The DNA of various insects could be preserved in acetone at room temperature in good condition for several years. In addition to the DNA of the host insects, the DNA of their endosymbionts, including Buchnera and other mycetocyte symbionts, Wolbachia, and gut bacteria, was amplified by PCR after several years of acetone storage. The RNA and protein of the pea aphid and its endosymbiont were also preserved for several years in acetone. After 2 years' storage in acetone, proteins of A. pisum could be analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting, and the endosymbiotic bacteria were successfully detected by immunohistochemistry and in situ hybridization on the tissue sections.
Fukatsu
T
T
National Institute of Bioscience and Human Technology, Agency of Industrial Science and Technology, Tsukuba, Ibaraki, Japan. fukatsu@nibh.go.jp
D000096:Acetone; D000818:Animals; D001042:Aphids / Q000235:genetics* / Q000382:microbiology*; D020635:Buchnera / Q000502:physiology*; D004247:DNA / Q000737:chemistry / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D000431:Ethanol; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification; D012313:RNA / Q000737:chemistry / Q000302:isolation & purification; D013048:Specimen Handling; D013559:Symbiosis; D014021:Tissue Preservation
D011506:Proteins; D000096:Acetone; D000431:Ethanol; D012313:RNA; D004247:DNA