Biology Reference
In-Depth Information
2.5 Microscopy
and Photography
Equipment
Microscopy and the acquired images are the most critical aspects
for success in obtaining quantitative morphological data using the
current protocol. It is essential to have the capacity to accurately
identify plants that are actively being silenced.
1. This method does not rely on expensive microscopy setups,
but can use high-end fl uorescence stereo microscopes, which
are available in many laboratories to observe small transgenic
animals and plants expressing GFP constructs. We have suc-
cessfully used two types of microscopes, a Leica MZ16FA with
a 1x lens and a Zeiss Discovery with a 1× lens. For routine
analysis, use a zoom that provides a magnifi cation where one
pixel is equivalent to 1
m. A lower level of magnifi cation is
not recommended, but higher magnifi cations may be used; for
this purpose we provide a calibration option in the accompa-
nying ImageJ macro.
2. A fi lter cube with a 480/40 bandpass excitation, a 505 long-
pass dichroic mirror, and a 510 longpass emission fi lter should
be used to allow for the visualization of GFP and chlorophyll
auto-fl uorescence simultaneously.
3. A color digital camera is recommended with a format size of at
least 1,000 × 1,000 pixels. A black and white digital camera
can also be used, but the active silencing status of the plant will
have to be separately recorded during analysis (see below).
μ
2.6 Images
The ImageJ macro provided is designed to work with a variety of
images. It is recommended to collect 12 bit/channel color images
and saving TIF stacks, with each image in the stack comprised of
one plant. The complete stack should correspond to a single “treat-
ment” with one class of RNAi construct. The macro will also work
with 8 bit/channel images (RGB) and 8-32 bit black and white
images (
see
Note 4
).
3
Methods
1. For the building of RNAi constructs, it is recommended to
use the vector pUGGi [
12
], which contains two inverted
Gateway cassettes. This allows for the easy design of plasmids
for the expression of hairpin-based RNAi constructs with a
single LR reaction. Sequences between 200 and 400 bp are
routinely used to silence a gene. When the degree of conserva-
tion is high between homologous genes, it is possible to use a
single construct directed to the coding sequence of one of the
genes to silence two or more genes [
5
]. For this type of multi-
gene silencing, the approximate cutoff in identity at the nucle-
otide level is 90 % between genes. When viable, you should
use this initial strategy for testing the participation of a gene
3.1 RNAi
Vector Design
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