Singer Lab Protocol:
In situ hybridization of mammalian cells (RNA and Oligonucleotide
probes)
Please reference
this web site (http://www.singerlab.org/protocols)
if this protocol contributes to your publication or presentation.
I.
General Considerations
-
In
situ hybridization is a powerful technique which can be very informative.
It should be however carefully controlled since it can lead to
artefacts. One of the most important considerations is fixation.
Formaldehyde is widely used as fixative. Ideally, fixation should
be strong enough to prevent loss of material during the hybridization
and washing steps, but mild enough to allow the probe to penetrate
everywhere in the cell. For this reason, DNA oligonucleotide probes
are preferable: they are short (a 50 base probe is 50 times smaller
than the Fab fragment of an antibody), which means that they penetrate
cells very well, can be washed under mild conditions, are compatible
with retention of intra-cellular RNA, are very stable and are
easily synthesized. Short RNA probes (50-100 nt; probes longer
than 200 nucleotides should be avoided) are similar to oligonucleotides,
but they necessitate more stringent washing conditions, and some
cellular RNA may be lost at this step since the formaldehyde fixation
does not resist temperatures well. The only advantage of RNA probes
over oligonucleotides is lower cost, unless many samples are being
processed (since one DNA synthesis produces enough probe for thousands
of samples.)
- When performing
in situ hybridization for the first time, it is often desirable
to monitor cytoplasmic RNA loss during the procedure by staining
the slides with propidium iodide during the steps in the procedure.
II. Fixation
-
Mammalian cells are grown directly onto coverslips,
washed once in 1X PBS (100 mM Na2HPO4,
20 mM KH2PO4,
137 mM NaCl, 27 mM KCl, pH 7.4), and fixed for 10 min at room
temperature in 4% formaldehyde (40% liquid stock purchased from
Electron Microscopy Science), 10% acetic acid, 1X PBS.
NB: The
presence of acetic acid improves detection of nuclear RNA.
It can be replaced by triton extraction prior to fixation. Neither
of those treatment is necessary when short (50 bases) oligonucleotides
probes are used (30 min fixation in 4% formaldehyde, 1X PBS is
sufficient in this case), or when one wants to detect cytoplasmic
RNAs.
- After
2 washes in PBS, cells are permeabilized by treatment with 70%
ethanol for at least overnight at 4C.
III.
Hybridization
- Cells are
rehydrated for 5 minutes at room temperature, in 2x SSC (300 mM
NaCl, 30 mM sodium citrate, pH 7.0), 50% formamide. Cells are
hybridized overnight at 37C in 40 µl of a mixture containing
10% dextran sulfate, 2 mM vanadyl-ribonucleoside complex, 0.02%
RNAse-free BSA, 40 µg E.coli tRNA, 2x SSC, 50% formamide,
30 ng of probe.
NB:
The amount of probe can be diminished 5 times without much
loss in signal in case background in the cells is high. Often
addition of a nonsense probe (N-50, where N is all nucleotides)
significantly reduces background.
-
For detection of poly(A) RNA using polydT(50), 15% formamide is
used at 37C. Only 5 ng of probe is necessary per sample;
hybridization times routinely take an hour, due to the low complexity
of the reactions.
IV.
Washing:
- Cells are
washed twice for 30 min at the appropriate stringency: 2x SSC,
50% formamide, 37C for oligonucleotide probes; 0.1x SSC, 50% formamide,
50C for RNA probes.
NB:
Washing conditions may be adjusted for each probe, and can be optimized
by Northern blotting. Probes that are labelled to a high specific
activity (see below) tend to give higher background, but addition
of 0.1% NP40 or 0.1% SDS in the washing buffer can diminish this
background.
V. RNAse treatment
(optional):
- RNase treatment
can remove background binding of the probes. It is however better
to avoid it when possible, since it leads to loss of RNA from
the cells. In most cases, it is possible to remove the background
by adjusting the stringency of washing and the amount of probe
in the hybridization mixture.
VI. Antibody detection
(optional):
- Digoxigenin-labeled
probes can be detected with sheep anti-digoxigenin antibodies
(1/200, Boehringer Mannheim), and then with donkey anti-sheep
antibodies conjugated to fluorescein (1/150, Sigma). Slides are
incubated for 1 hour at 37C in 2x SSC, 8% formamide, 2 mM vanadyl-ribonucleoside
complex, 0.2% RNAse-free BSA, and washed twice for 15 min in 2x
SSC, 8% formamide at room temperature.
NB:
Fluorescent antibodies tend to give a high background, which is
efficiently removed by the presence of 8% formamide in the incubation
buffer.
VII.
Mounting:
- Slides
are washed in 1X PBS, and mounted in 90% glycerol, PBS, 1 mg/ml
p-phenylendiamine, 0.1 µg/ml DAPI.
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