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Metagenomic Analysis
of Thermal and Saline Environments
Your Data: An RNAlater® Story
RNAlater® is an aqueous, non-toxic
tissue storage reagent that stabilizes and protects cellular
RNA in intact, unfrozen tissue samples. RNAlater eliminates
the need to immediately process tissue samples or to freeze samples
in liquid nitrogen for later processing. Tissue pieces can be
harvested and submerged in RNAlater for storage, without
jeopardizing the quality or quantity of RNA obtained after subsequent
RNA isolation. This characteristic of RNAlater was critical
for the collection of algal samples in remote areas of China
in the research story provided here by Dr. Shaun Heaphy and colleagues.
Metagenomic Analysis of Thermal and Saline Environments
in China
Dr. Shaun Heaphy, Dr. S. Grant, and Prof. William
Grant
Department of Infection, Immunity, & Inflammation
Medical School University of Leicester, Leicester, LE1 9HN, England
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Figure
1. Collecting Algal Mat Samples
in Remote China. Bill Grant and Shaun Heaphy
(center) collect an algal mat sample from a geothermal
pool, Long Pu, in P.R. China while a colleague looks
on. Samples were immediately immersed in 10 volumes
RNAlater® Tissue Collection:RNA Storage
Solution at ambient temperature and carried out of
the field. This and similar samples were later used
for RNA purification and cDNA library construction.
The sampling trips were made possible by the support
of the EU (QLK3-CT-2002-01972) and the Institute of
Microbiology, Chinese Academy of Sciences.
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Well established protocols can be used for
direct isolation of DNA from prokaryotic environmental samples
to construct metagenomic libraries. Such libraries can be searched
for novel proteins with useful biological activities without
the need for prior cultivation or identification of the source
organism. However, similar metagenomic ‘gene mining’ in
eukaryotic DNA with a more widespread presence of introns, requires
the construction of cDNA libraries from mRNA in the samples.
It is therefore essential that the RNA in the samples be stabilized
immediately following collection. To this end, this group investigated
the usefulness of RNAlater Tissue Collection:RNA Storage
Solution in preventing RNA degradation in samples of algal mats
from geothermal areas and salt lakes in P.R. China. The sampling
locations were frequently in remote regions with limited access
to refrigeration, so the ability to stabilize the samples at
ambient temperature was crucial to the project (Figure 1).
Prior to the expedition, preliminary tests
were carried out on known species of bacteria, fungi, and protozoa,
and on an environmental sample, activated sludge from a local
sewage treatment plant. Samples were collected and stored in
RNAlater at ambient temperature or -20°C for several
weeks. Total RNA purified from the samples appeared undegraded
when stored under these conditions. Lambda cDNA libraries were
later successfully constructed from total RNA and from mRNA extracted
from the environmental samples gathered in China and stored similarly.
Eukaryotic ORFS were readily identified. Read more about this
fascinating research account in:
Grant S, Grant W, Cowan D, Jones
B, Ma Y, Ventosa A, Heaphy S. Identification of Open Reading
Frames in Metagenomic cDNA Libraries made from Environmental
Samples. Appl Environ Microbiol (in press).
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