3.5 Quantifying RNA
Movement
The following section describes how to quantify RNA movement
using the PVX movement assay. For effective monitoring of movement, the ROI-containing viral RNA should be tagged to distinguish it from native, plant-encoded RNA (Fig. 3). RT-qPCR
reactions with gene-specific primers are utilized to score RNA levels
in both the source leaf and the target organ. Control and test
reactions will provide quantitative data on the movement of viral
RNA with and without the ROI. Results may be interpreted with a
simple statistical test for significance (see Fig. 2 for an example).
1. Clone and tag transcribed RNAs to be inoculated with a unique
sequence to distinguish them from native transcripts (see Notes
9 and 10; Fig. 3).
2. Perform inoculation, harvest of samples, and RNA extraction
as described in Subheading 3.4, steps 1–11. In addition to
RNA samples from plants inoculated with the ROI-expressing
virus, RNA samples should include control samples derived
from plants inoculated with (1) water, (2) PVX vector with its
coat protein (mobile), and (3) the PVX vector with no coat
protein and without the ROI (nonmobile).
3. Treat two micrograms of each RNA samples with RNase-free
DNase (see Subheading 3.1, steps 2–13).
4. Set up RT-qPCR reactions by combining 50 ng of total
DNA-free RNA sample with 7.5 μL of One-Step SYBR Green
Master Mix, 0.3 μL of qScript One-Step Reverse Transcriptase,
and target-specific primers (200 nM) in a total volume of
15 μL.
5. Perform qPCR reactions with a real-time PCR thermocycler
(see Note 11). All reactions should be performed in triplicate
with internal standards for normalization (detection of RNA
transcripts of “housekeeping” genes, such as Actin8 or
GAPDH using gene-specific primers). Fast quantitative PCR
cycling parameters for complementary DNA (cDNA) synthesis
are 50
C, 5 min. Taq activation: 95
C for 2 min, followed by
40 cycles of 95
C for 15 s and 60
C for 30 s. Conditions for
qPCR may vary slightly depending on the primer design and
the internal standard employed.
6. Check PCR specificity using melting curve analysis and calculate RNA values as the 2
ÀΔΔCt value relative to the mean values
of control samples [27]. Obtain standard deviations of the
means of three biological replicates with two technical replicates and implement statistical analysis using the Student’s ttest using GraphPad Prism (6.0 version) [28].
PVX-Based RNA Movement Assay
191
Movement
The following section describes how to quantify RNA movement
using the PVX movement assay. For effective monitoring of movement, the ROI-containing viral RNA should be tagged to distinguish it from native, plant-encoded RNA (Fig. 3). RT-qPCR
reactions with gene-specific primers are utilized to score RNA levels
in both the source leaf and the target organ. Control and test
reactions will provide quantitative data on the movement of viral
RNA with and without the ROI. Results may be interpreted with a
simple statistical test for significance (see Fig. 2 for an example).
1. Clone and tag transcribed RNAs to be inoculated with a unique
sequence to distinguish them from native transcripts (see Notes
9 and 10; Fig. 3).
2. Perform inoculation, harvest of samples, and RNA extraction
as described in Subheading 3.4, steps 1–11. In addition to
RNA samples from plants inoculated with the ROI-expressing
virus, RNA samples should include control samples derived
from plants inoculated with (1) water, (2) PVX vector with its
coat protein (mobile), and (3) the PVX vector with no coat
protein and without the ROI (nonmobile).
3. Treat two micrograms of each RNA samples with RNase-free
DNase (see Subheading 3.1, steps 2–13).
4. Set up RT-qPCR reactions by combining 50 ng of total
DNA-free RNA sample with 7.5 μL of One-Step SYBR Green
Master Mix, 0.3 μL of qScript One-Step Reverse Transcriptase,
and target-specific primers (200 nM) in a total volume of
15 μL.
5. Perform qPCR reactions with a real-time PCR thermocycler
(see Note 11). All reactions should be performed in triplicate
with internal standards for normalization (detection of RNA
transcripts of “housekeeping” genes, such as Actin8 or
GAPDH using gene-specific primers). Fast quantitative PCR
cycling parameters for complementary DNA (cDNA) synthesis
are 50
C, 5 min. Taq activation: 95
C for 2 min, followed by
40 cycles of 95
C for 15 s and 60
C for 30 s. Conditions for
qPCR may vary slightly depending on the primer design and
the internal standard employed.
6. Check PCR specificity using melting curve analysis and calculate RNA values as the 2
ÀΔΔCt value relative to the mean values
of control samples [27]. Obtain standard deviations of the
means of three biological replicates with two technical replicates and implement statistical analysis using the Student’s ttest using GraphPad Prism (6.0 version) [28].
PVX-Based RNA Movement Assay
191