Biomedical Engineering Reference
In-Depth Information
probes permits evaluation of specific metabolic features that may be shared by unrelated
organisms, where group-specific probes cannot capture the breadth of metabolic diversity.
In general, RNA coding for specific metabolic processes is present at lower concentrations
than ribosomal RNA. These lower concentrations result in decreased fluorescent signal inten-
sity and often complicate functional FISH analyses.
The use of FISH for direct examination via fluorescent microscopy requires several
steps: (1) fixation of cells on a filter or microscope slide, (2) hybridization of probes with target
genes, (3) washing of excess probe material, and (4) microscopic evaluation. Slight variations
in the specifics associated with these steps occur depending if the analysis is performed on
planktonic or biofilm communities, or if concentration of cells (by filtration or centrifugation)
from larger sample volumes is required. However, the general principles and processes remain
consistent regardless of the origin of the sample. The initial fixation step utilizes a number of
chemicals for attaching the cells onto a glass slide, then drying them in place. Commonly, dilute
paraformaldehyde is used for “fixing” the cells to the slide, followed by baths in successively
higher concentrations of ethanol for the drying step. This process kills the cells, but preserves
the enzymes, RNA and DNA, thus providing a “snapshot” of current metabolic activity. After
fixation, the cell walls and membranes become more permeable and diffusion of small
molecules (such as FISH probes) becomes possible. During the hybridization step, the cells
are bathed in a solution containing a buffering reagent, the probe (or mixture of several
different probes), and variable concentrations of formamide. Formamide affects the stability
of probe-RNA duplexes and allows incubation at elevated temperatures (35-50
C), thereby
improving the hybridization reaction kinetics. The formamide concentration used depends
upon the nucleotide composition of the probe and the probe hybridization stringency desired.
During this hybridization step, FISH probes diffuse through the cell membrane and bind with
complementary sections of RNA. The temperatures, although elevated, are not significant
enough to denature DNA, thus limited binding occurs. Nonspecific fluorescent counter-stains
that intercalate within DNA, such as 4
0
,6-diamidino-2-phenylindole (DAPI), are sometimes
added during this step to provide a method of determining total cell numbers versus those
with the targeted gene of interest. After hybridization and incubation, the cells undergo a
washing step using a dilute salt solution to remove unbound probe and disrupt any nonspecific
hybridization. Varying the salt concentration and wash temperature also provides another
control over the stringency of hybridization.
Examination of the fluorescently labeled cells requires a microscope equipped with an
ultraviolet bulb or lasers emitting specific wavelengths. Since different fluorophores adsorb
and emit at a variety of wavelengths, multiple probes may be used on a single sample and a
variety of target cells screened at once (e.g.,
Dhc
cells and sulfate-reducing microbes). Cell
concentrations are determined through manual or automated cell counting, then back-
calculated to a cells per unit volume (or mass, in the case of biofilms). Thus, the native
concentration of cells containing the targeted gene of interest can be estimated for environ-
mental samples.
6.6.3 Limitations
The limitations of FISH reflect those discussed for qPCR in the previous section for many
of the same reasons. Sample selection must be representative, although since no amplification
step occurs, this is less critical for FISH than for qPCR. Instead, probe selection and an
understanding of probe specificity becomes even more crucial when using FISH. However,
sample variability (e.g., differing matrices) can result in higher natural background fluores-
cence, which makes the sensitivity of FISH difficult to assess. Probe-RNA hybridization
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