4 Notes
1. Kits may be purchased from Qiagen, Promega, Ambion, Biolabs, Invitrogen, or any other numerous commercial kits available. We used the Qiagen Plant RNeasy Purification Kit.
2. Add each of the individual components as described in this
protocol. There are in vitro transcription kits available from
different companies. We assemble reaction components using
reagents purchased from Biolabs and Promega, which are less
expensive.
3. This step is important to maintain high humidity to increase
seed germination.
4. Avoid applying too much carborundum on leaves. Excessive
contact can lead to severe damage of leaves after finger-rubbing
them during inoculation.
5. This minimizes desiccation and enhances the inoculation.
6. Plant material may be stored at À80
C for later use.
7. Leaf inoculation also works well on potato cultivars.
8. For RT-PCR, kits may be purchased from Qiagen, Promega,
Ambion, Biolabs, Invitrogen, or any other company. We commonly use the One-Step RT-PCR kit from Promega or the
Next High-Fidelity 2Â PCR Master Mix from NEB. Using
the same commercial kit for all RT-PCR assays ensures consistency and quality control.
9. See Table S5 in the Supplemental files of [26] for examples of
primers designed for the quantitative analysis of a mobile RNA,
StBEL5, and an immobile negative control, StBEL14, by
RT-qPCR.
10. In the example shown in Fig. 2, reverse primers for creation of
the StBEL constructs were designed to include a histidine-tag
sequence for specific ROI detection (Fig. 3).
11. For RT-qPCR, we commonly use the qScript One-Step SYBR
Green qRT-PCR kit (Quanta Biosciences) with the Eco RealTime PCR thermocycler (Illumina). However, any other
RT-qPCR real-time system may be used.
Acknowledgments
This work was in part funded by Ministry of Science and Technology of China (National Key R&D Program 2017YFE0110900),
Ministry of Agriculture of China (National Transgenic Program
2016ZX08009001-004), the National Natural Science Foundation of China (31872636, 31370180), Zhejiang Provincial Natural
192
Zhiming Yu et al.
1. Kits may be purchased from Qiagen, Promega, Ambion, Biolabs, Invitrogen, or any other numerous commercial kits available. We used the Qiagen Plant RNeasy Purification Kit.
2. Add each of the individual components as described in this
protocol. There are in vitro transcription kits available from
different companies. We assemble reaction components using
reagents purchased from Biolabs and Promega, which are less
expensive.
3. This step is important to maintain high humidity to increase
seed germination.
4. Avoid applying too much carborundum on leaves. Excessive
contact can lead to severe damage of leaves after finger-rubbing
them during inoculation.
5. This minimizes desiccation and enhances the inoculation.
6. Plant material may be stored at À80
C for later use.
7. Leaf inoculation also works well on potato cultivars.
8. For RT-PCR, kits may be purchased from Qiagen, Promega,
Ambion, Biolabs, Invitrogen, or any other company. We commonly use the One-Step RT-PCR kit from Promega or the
Next High-Fidelity 2Â PCR Master Mix from NEB. Using
the same commercial kit for all RT-PCR assays ensures consistency and quality control.
9. See Table S5 in the Supplemental files of [26] for examples of
primers designed for the quantitative analysis of a mobile RNA,
StBEL5, and an immobile negative control, StBEL14, by
RT-qPCR.
10. In the example shown in Fig. 2, reverse primers for creation of
the StBEL constructs were designed to include a histidine-tag
sequence for specific ROI detection (Fig. 3).
11. For RT-qPCR, we commonly use the qScript One-Step SYBR
Green qRT-PCR kit (Quanta Biosciences) with the Eco RealTime PCR thermocycler (Illumina). However, any other
RT-qPCR real-time system may be used.
Acknowledgments
This work was in part funded by Ministry of Science and Technology of China (National Key R&D Program 2017YFE0110900),
Ministry of Agriculture of China (National Transgenic Program
2016ZX08009001-004), the National Natural Science Foundation of China (31872636, 31370180), Zhejiang Provincial Natural
192
Zhiming Yu et al.