Simultaneous recovery of RNA and DNA from soils and sediments.
Recovery of mRNA from environmental samples for measurement of in situ metabolic activities is a significant challenge. A robust, simple, rapid, and effective method was developed for simultaneous recovery of both RNA and DNA from soils of diverse composition by adapting our previous grinding-based cell lysis method (Zhou et al., Appl. Environ. Microbiol. 62:316-322, 1996) for DNA extraction. One of the key differences is that the samples are ground in a denaturing solution at a temperature below 0 degrees C to inactivate nuclease activity. Two different methods were evaluated for separating RNA from DNA. Among the methods examined for RNA purification, anion exchange resin gave the best results in terms of RNA integrity, yield, and purity. With the optimized protocol, intact RNA and high-molecular-weight DNA were simultaneously recovered from 19 soil and stream sediment samples of diverse composition. The RNA yield from these samples ranged from 1.4 to 56 microg g of soil(-1) dry weight), whereas the DNA yield ranged from 23 to 435 microg g(-1). In addition, studies with the same soil sample showed that the DNA yield was, on average, 40% higher than that in our previous procedure and 68% higher than that in a commercial bead milling method. For the majority of the samples, the DNA and RNA recovered were of sufficient purity for nuclease digestion, microarray hybridization, and PCR or reverse transcription-PCR amplification.
Hurt
R A
RA
Environmental Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 38831, USA.
Qiu
X
X
Wu
L
L
Roh
Y
Y
Palumbo
A V
AV
Tiedje
J M
JM
Zhou
J
J
D004269:DNA, Bacterial / Q000302:isolation & purification*; D004271:DNA, Fungal / Q000302:isolation & purification; D019015:Geologic Sediments / Q000737:chemistry* / Q000382:microbiology; D020411:Oligonucleotide Array Sequence Analysis; D016133:Polymerase Chain Reaction; D012329:RNA, Bacterial / Q000302:isolation & purification*; D012331:RNA, Fungal / Q000302:isolation & purification; D012333:RNA, Messenger / Q000302:isolation & purification; D012987:Soil / Q000032:analysis*; D012988:Soil Microbiology
D004269:DNA, Bacterial; D004271:DNA, Fungal; D012329:RNA, Bacterial; D012331:RNA, Fungal; D012333:RNA, Messenger; D012987:Soil