5. Add 5 μL of 20 mM GTP.
6. Incubate at 37
C for 35 min.
7. Add 45 μL of 10 mM Tris, pH 8.5, to the mixture.
8. Purify the in vitro-transcribed RNA by following steps 5
through 12 of the protocol described under Subheading 3.1.
9. Dissolve the RNA in 40 μL RNase-free water (pH 7.0). RNA is
now ready for plant inoculation.
3.4 Plant Inoculation
and Molecular
Analysis of RNA LongDistance Trafficking
Using RT-PCR
This section describes the inoculation of plant leaves with the
in vitro-transcribed RNA and analysis of transported RNA by
RNA isolation and RT-PCR.
1. Sow Nicotiana benthamiana seeds on soil in 6-in. plastic pots.
2. Cover the soil with transparent film (see Note 3).
3. Germinate the seeds in a growth chamber with the temperature
at 25
C under 16-h light/8-h dark and allow plantlets to grow
for approximately 14 days.
4. Transfer individual plantlets to individual pots and keep plants
growing under the same conditions.
5. At the six-leaf stage and with plants still in the growth chamber,
apply a thin layer of fine carborundum onto two small leaves
(see Note 4).
6. Drop 5 μL of RNA transcripts prepared by in vitro transcription
(see Subheading 3.3) onto each of the two carborundumdusted small leaves.
7. Wear clean gloves and gently rub the RNA droplet across the
entire laminar of the leaves.
8. Spray water onto the inoculated plants (see Note 5).
9. Incubate plants in a growth chamber at 25
C under 16-h
light/8-h dark for 7–14 days.
10. Collect the inoculated leaves and samples of target organs for
RNA extraction (see Note 6). Pool samples from three independent plants each of (1) inoculated leaves and other organs
and (2) the same leaves of non-inoculated or buffer-inoculated
control plants. Repeat steps 7–12 including tissue sampling
two times to create samples from a total of three biological
replicate experiments (see Note 7).
11. Extract RNAs from samples using a commercial kit following
the manufacturer’s protocol (see Note 1).
12. Use 150 ng total RNA from each sample for RT-PCR assays
using a commercial kit (see Note 8).
13. Using a UV light source, visualize the amplified products
resolved by agarose gel electrophoresis through a 1.2% agarose
gel in TBE buffer (Figs. 1b, c, and 2a).
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Zhiming Yu et al.
6. Incubate at 37
C for 35 min.
7. Add 45 μL of 10 mM Tris, pH 8.5, to the mixture.
8. Purify the in vitro-transcribed RNA by following steps 5
through 12 of the protocol described under Subheading 3.1.
9. Dissolve the RNA in 40 μL RNase-free water (pH 7.0). RNA is
now ready for plant inoculation.
3.4 Plant Inoculation
and Molecular
Analysis of RNA LongDistance Trafficking
Using RT-PCR
This section describes the inoculation of plant leaves with the
in vitro-transcribed RNA and analysis of transported RNA by
RNA isolation and RT-PCR.
1. Sow Nicotiana benthamiana seeds on soil in 6-in. plastic pots.
2. Cover the soil with transparent film (see Note 3).
3. Germinate the seeds in a growth chamber with the temperature
at 25
C under 16-h light/8-h dark and allow plantlets to grow
for approximately 14 days.
4. Transfer individual plantlets to individual pots and keep plants
growing under the same conditions.
5. At the six-leaf stage and with plants still in the growth chamber,
apply a thin layer of fine carborundum onto two small leaves
(see Note 4).
6. Drop 5 μL of RNA transcripts prepared by in vitro transcription
(see Subheading 3.3) onto each of the two carborundumdusted small leaves.
7. Wear clean gloves and gently rub the RNA droplet across the
entire laminar of the leaves.
8. Spray water onto the inoculated plants (see Note 5).
9. Incubate plants in a growth chamber at 25
C under 16-h
light/8-h dark for 7–14 days.
10. Collect the inoculated leaves and samples of target organs for
RNA extraction (see Note 6). Pool samples from three independent plants each of (1) inoculated leaves and other organs
and (2) the same leaves of non-inoculated or buffer-inoculated
control plants. Repeat steps 7–12 including tissue sampling
two times to create samples from a total of three biological
replicate experiments (see Note 7).
11. Extract RNAs from samples using a commercial kit following
the manufacturer’s protocol (see Note 1).
12. Use 150 ng total RNA from each sample for RT-PCR assays
using a commercial kit (see Note 8).
13. Using a UV light source, visualize the amplified products
resolved by agarose gel electrophoresis through a 1.2% agarose
gel in TBE buffer (Figs. 1b, c, and 2a).
190
Zhiming Yu et al.