virus vector using RT-PCR. Upon linearization, the RNA is synthesized to large scale by using in vitro transcription. The RNA of
interest is then incorporated into tobacco leaves through direct
inoculation to be tested for mobility. Gel-based analysis of the
RT-PCR product can be used to confirm movement of any RNAs
of interest (Fig. 1). Quantitative RT-PCR (RT-qPCR) is utilized to
monitor RNA movement or test elements that mediate movement.
This simple protocol may be used for any RNA sequence of interest
to assess its capacity for long-distance transport from source leaves
to distal target organs.
2 Materials
Prepare all solutions using high-purity deionized water and analytical grade reagents. Prepare and store all reagents at room temperature unless otherwise noted. Follow all safety regulations when
disposing of waste or biohazardous materials.
2.1 Plasmid
Preparation
1. Sterile distilled water.
2. RNase-free water.
3. Bovine serum albumin (BSA).
4. Phenol.
5. Chloroform.
6. 3.0 M NaAc (sodium acetate) pH 5.2.
7. PVX/CP vector (obtained from Yiguo Hong).
8. PVX/ΔCP vector (obtained from Yiguo Hong).
9. MluI enzyme (10 units/μL).
10. MluI 1Â buffer: 100 mM NaCl, 50 mM Tris–HCl, 10 mM
MgCl 2 100 μg/mL BSA (pH 7.9).
11. EcoRV enzyme (20 units/μL).
12. EcoRV 1Â buffer: 100 mM NaCl, 50 mM Tris–HCl, 10 mM
MgCl 2 100 μg/mL BSA (pH 7.9).
13. E. coli strain DH5α.
2.2 In Vitro
Transcription and
RT-qPCR
1. Total RNA extraction kit.
2. RNase-free DNase: 30 Kunitz units per RNA prep.
3. 10Â DNase buffer: 100 mM Tris–HCl, 25 mM MgCl 2 ,
5.0 mM CaCl 2 (pH 7.6).
4. 70% Ethanol.
5. 100% Ethanol.
6. One-Step SYBR Green Master Mix.
7. qScript One-Step Reverse Transcriptase.
PVX-Based RNA Movement Assay
185
interest is then incorporated into tobacco leaves through direct
inoculation to be tested for mobility. Gel-based analysis of the
RT-PCR product can be used to confirm movement of any RNAs
of interest (Fig. 1). Quantitative RT-PCR (RT-qPCR) is utilized to
monitor RNA movement or test elements that mediate movement.
This simple protocol may be used for any RNA sequence of interest
to assess its capacity for long-distance transport from source leaves
to distal target organs.
2 Materials
Prepare all solutions using high-purity deionized water and analytical grade reagents. Prepare and store all reagents at room temperature unless otherwise noted. Follow all safety regulations when
disposing of waste or biohazardous materials.
2.1 Plasmid
Preparation
1. Sterile distilled water.
2. RNase-free water.
3. Bovine serum albumin (BSA).
4. Phenol.
5. Chloroform.
6. 3.0 M NaAc (sodium acetate) pH 5.2.
7. PVX/CP vector (obtained from Yiguo Hong).
8. PVX/ΔCP vector (obtained from Yiguo Hong).
9. MluI enzyme (10 units/μL).
10. MluI 1Â buffer: 100 mM NaCl, 50 mM Tris–HCl, 10 mM
MgCl 2 100 μg/mL BSA (pH 7.9).
11. EcoRV enzyme (20 units/μL).
12. EcoRV 1Â buffer: 100 mM NaCl, 50 mM Tris–HCl, 10 mM
MgCl 2 100 μg/mL BSA (pH 7.9).
13. E. coli strain DH5α.
2.2 In Vitro
Transcription and
RT-qPCR
1. Total RNA extraction kit.
2. RNase-free DNase: 30 Kunitz units per RNA prep.
3. 10Â DNase buffer: 100 mM Tris–HCl, 25 mM MgCl 2 ,
5.0 mM CaCl 2 (pH 7.6).
4. 70% Ethanol.
5. 100% Ethanol.
6. One-Step SYBR Green Master Mix.
7. qScript One-Step Reverse Transcriptase.
PVX-Based RNA Movement Assay
185