cellular resolution. To analyze mRNA distribution with a subcellular resolution, (3) a sensitive fluorescence detection system based
on the enzymatic activity of a horseradish peroxidase (HRP) can be
used. In combination with a tyramide signal amplification (TSA)
system, the enzyme catalyzes multiple fluorescent labeling of the
antibody. This enables to detect specific RNAs including mRNAs
and small noncoding RNAs with high sensitivity and to determine
their location with resolution at the subcellular level [3].
The enzymatic signal amplification achieved by the alkaline
phosphatase or the peroxidase is essential for mRNA visualization
in whole tissues, especially if cells will be analyzed deeper in the
tissue. The fluorescent signal emitted by directly fluorescentlabeled RNA probes or fluorescent-labeled antibodies is often too
weak and can commonly only be visualized in close proximity to the
cover slide.
In conclusion, we recommend to use AP with NBT/BCIP for
general expression studies and the HRP with TSA for the analysis of
subcellular RNA distribution. The presented protocols are based on
[4, 5] with modifications. A workflow overview can be found in
Fig. 2. Variations of the protocols optimized for germline cells,
ovules, and developing seeds have been published elsewhere [6].
2 Materials
2.1 Labware
1. Pipettes.
2. PCR machine.
3. Agarose gel electrophoresis equipment.
4. Thermoshaker.
5. Benchtop centrifuge.
6. Plant growth chamber.
7. Sterilization oven.
8. Bottles with thermostable (!200
C) screw cap and pouring
rings.
9. Razor blade.
10. Tweezers.
11. Syringe cannulas.
12. Vacuum concentrator.
13. Sieves to carry tissue samples for liquid exchange (see Note 1).
14. Glass petri dishes.
15. Glass Pasteur pipettes.
16. Shaker.
17. Sterile multi-well plates (fitting to the sieve size).
RNA-FISH
5
on the enzymatic activity of a horseradish peroxidase (HRP) can be
used. In combination with a tyramide signal amplification (TSA)
system, the enzyme catalyzes multiple fluorescent labeling of the
antibody. This enables to detect specific RNAs including mRNAs
and small noncoding RNAs with high sensitivity and to determine
their location with resolution at the subcellular level [3].
The enzymatic signal amplification achieved by the alkaline
phosphatase or the peroxidase is essential for mRNA visualization
in whole tissues, especially if cells will be analyzed deeper in the
tissue. The fluorescent signal emitted by directly fluorescentlabeled RNA probes or fluorescent-labeled antibodies is often too
weak and can commonly only be visualized in close proximity to the
cover slide.
In conclusion, we recommend to use AP with NBT/BCIP for
general expression studies and the HRP with TSA for the analysis of
subcellular RNA distribution. The presented protocols are based on
[4, 5] with modifications. A workflow overview can be found in
Fig. 2. Variations of the protocols optimized for germline cells,
ovules, and developing seeds have been published elsewhere [6].
2 Materials
2.1 Labware
1. Pipettes.
2. PCR machine.
3. Agarose gel electrophoresis equipment.
4. Thermoshaker.
5. Benchtop centrifuge.
6. Plant growth chamber.
7. Sterilization oven.
8. Bottles with thermostable (!200
C) screw cap and pouring
rings.
9. Razor blade.
10. Tweezers.
11. Syringe cannulas.
12. Vacuum concentrator.
13. Sieves to carry tissue samples for liquid exchange (see Note 1).
14. Glass petri dishes.
15. Glass Pasteur pipettes.
16. Shaker.
17. Sterile multi-well plates (fitting to the sieve size).
RNA-FISH
5