Biology Reference
In-Depth Information
7. To help appreciate the statistical signifi cance of the differences,
plot the mean normalized area and circularity values with the
standard error of the mean using a logarithmic scale. The
mean solidity values should be plotted with the standard error
of the mean on a linear scale.
3.8 Verifi cation
of RNAi Activity and
Expression of Protein
Sometimes it may be necessary to verify that RNAi constructs are
active by a more direct indicator than only the internal nuclear
GFP-GUS reporter. This is particularly important in situations
where a phenotype is not observed. Two approaches can be used
to address the level of silencing attained: reverse transcription fol-
lowed by quantitative PCR (qRT-PCR), or immunofl uorescence.
We have used both methods successfully, but they both require a
considerable amount of effort, in particular, in order to isolate the
actively silencing plants from the background (
see
Note 15
). The
details for this process are beyond the scope of this chapter, but the
details to perform these procedures are detailed in the following
publications, for immunostaining [
3
,
5
] and for qRT-PCR [
6
,
7
].
4
Notes
1. Moss transformation can also be achieved by other means such
as particle bombardment and
Agrobacterium
[
15
-
18
], and
combined with protoplasting and regeneration, it may be pos-
sible to adapt these modes of transformation to this protocol.
2. Another very detailed protocol for culture and transformation
has also been recently published in this series [
19
]; this proto-
col can be used for transient transformation and should yield
very similar results.
3. Sometimes the maxi-preps will have very reduced yield. We
believe this is due to the use of ampicillin for selection, which
can result in the loss of plasmid in some bacterial cultures. We
routinely test an aliquot of the maxi-prep culture with a quick
mini-prep to determine that the plasmid concentration is
within the expected range for the kit.
4. The nomenclature in ImageJ can be confusing because multi-
channel color images are referred as RGB stacks, to differenti-
ate them from the conventional RGB images. A TIF stack
refers to a series of images in the TIF format and can be 8, 16,
or 32 bit. It is possible to have a TIF stack made of RGB
stacks, which is the case for TIF stacks of 12 bit/channel color
images. Further confusing the nomenclature, most cameras
have a 12 bit output which is interpreted by ImageJ as 16 bit
image; this is because the 12 bit data is contained within the
16 bit fi le size.
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