Poly(A) polymerase modification and reverse transcriptase PCR amplification of environmental RNA.
We describe a combination of two established techniques for a novel application for constructing full-length cDNA clone libraries from environmental RNA. The cDNA was cloned without the use of prescribed primers that target specific genes, and the procedure did not involve random priming. Purified RNA was first modified by addition of a poly(A) tail and then was amplified by using a commercially available reverse transcriptase PCR (RT-PCR) cDNA synthesis kit. To demonstrate the feasibility of this approach, a cDNA clone library was constructed from size-fractionated RNA (targeting 16S rRNA) purified from a geothermally heated soil in Yellowstone National Park in Wyoming. The resulting cDNA library contained clones representing Bacteria and Eukarya taxa and several mRNAs. There was no exact clone match between this library and a separate cDNA library generated from an RT-PCR performed with unmodified rRNA and Bacteria-specific forward and universal reverse primers that were designed from cultivated organisms; however, both libraries contained representatives of the Firmicutes and the alpha-Proteobacteria. Unexpectedly, there were no Archaea clones in the library generated from poly(A)-modified RNA. Additional RT-PCRs performed with universal and Archaea-biased primers and unmodified RNA demonstrated the presence of novel Archaea in the soil. Experiments with pure cultures of Sulfolobus solfataricus and Halobacterium halobium revealed that some Archaea rRNA may not be a suitable substrate for the poly(A) tail modification step. The protocol described here demonstrates the feasibility of directly accessing prokaryote RNA (rRNA and/or mRNA) in environmental samples, but the results also illustrate potentially important problems.
Botero
Lina M
LM
Thermal Biology Institute, Montana State University, Bozeman, MT 59717, USA.
D'Imperio
Seth
S
Burr
Mark
M
McDermott
Timothy R
TR
Young
Mark
M
Hassett
Daniel J
DJ
D001105:Archaea / Q000235:genetics / Q000302:isolation & purification; D001419:Bacteria / Q000235:genetics / Q000302:isolation & purification; D001483:Base Sequence; D004783:Environmental Microbiology; D019143:Evolution, Molecular; D015723:Gene Library; D010802:Phylogeny; D011062:Polynucleotide Adenylyltransferase; D019642:RNA, Archaeal / Q000235:genetics* / Q000302:isolation & purification; D012329:RNA, Bacterial / Q000235:genetics* / Q000302:isolation & purification; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012988:Soil Microbiology
D019642:RNA, Archaeal; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S; D011062:Polynucleotide Adenylyltransferase