Experiments

Aim

To prepare carrot agar media for the growth of fungal cultures.

Materials Required

1. Fresh carrots
2. Blender
3. Beaker
4. Muslin cloth
5. Agar powder
6. Vegetable peeler
7. Sterile water
8. Autoclave

Procedure

1. Take 200 g of carrots, wash and peel them.
2. Dice/chop the carrots and blend them.
3. Add 750 mL of sterile water to a beaker and pour the puree from the blender into the beaker.
4. Let the puree boil till the solution becomes clear. Discard any scum that floats to the top.
5. Sieve the cooled solution through a muslin cloth and add sterile water until the final volume is 1L.
6. Add 1.8% w/v (18 g) agar powder to the solution.
7. Autoclave the solution and pour it into petri plates.

Aim

Subculturing of Phytophthora capsici on carrot media petri plates.

Materials Required

1. Phytophthora capsici culture
2. Carrot agar petri plates
3. Laminar Hood
4. Inoculation loop
5. Forceps
6. Parafilm
7. 8 mm Cork borer
8. 70% Ethanol

Procedure

Protocol adopted from Mohsan et al. (2017b).

1. Prepare fresh carrot agar petri plates using carrot extract, agar, and sterile distilled water. Autoclave and pour it onto petri dishes; allow to solidify.
2. In a sterile laminar airflow chamber, sterilize the cork borer (dip the cutting edge in 70% ethanol, dry it properly, pass it through the flame, and let it cool). Take the fresh Phytophthora capsici culture (we sourced our culture from ICAR-Indian Institute of Spices Research, Kozhikode). Hold the cork borer vertically over the growing end of the fungal colony & press it firmly into the agar. Twist slightly to cut a circular plug. Sterilize the cork borer in between uses using the flame.
3. Transfer the plugs onto a fresh carrot agar plate (use forceps and an inoculation loop to aid the transfer); place the plugs with the mycelium side facing down. Parafilm the plate.
4. Incubate plates in the dark at 28°C.
5. After 4 to 5 days, mycelia would have colonized the whole plate, appearing as a mat, off-white in color.

References

Mohsan, M., Ali, S., Shahbaz, M. U., Saeed, S., Burhan, M., & Shahbaz, M. (2017). In vitro efficacy of different growth media and crude plant extracts against mycelial growth of Phytophthora capsici. Journal of Applied Biology & Biotechnology. https://doi.org/10.7324/jabb.2017.50407

Aim

To measure and visualize the growth of Phytophthora capsici by measuring colony diameter over time.

Materials Required

1. P. capsici culture
2. Carrot agar petri plates
3. Cork borer (8 mm diameter)
4. Forceps
5. Inoculation loop
6. 70% ethanol
7. Parafilm
8. Ruler/Vernier caliper

Procedure

Protocol adopted from (Lu et al., 2011c; Santos et al., 2023d; Z. Wang et al., 2009c).

1. Prepare fresh carrot agar petri plates using carrot extract, agar, and sterile distilled water. Autoclave and pour onto petri dishes, allowing them to solidify.
2. In a sterile laminar airflow chamber, sterilize the cork borer (dip the cutting edge in 70% ethanol, let it dry completely; then pass it through flame and let it cool). Take the fresh fungal culture. Hold the cork borer vertically over the growing end of the fungal colony & press it firmly into the agar. Twist slightly to cut a circular plug. Sterilize the cork borer in between uses using the flame.
3. Transfer a single plug onto a fresh carrot agar plate (use forceps and an inoculation loop to aid its transfer); place the plug with the mycelium side facing down in the center. Parafilm the plate. Prepare replicates to validate.
4. Incubate plates in the dark at 28°C.
5. Remove plates from incubation at set intervals (every 24/48 hrs). Measure the diameter of the colony in two perpendicular directions using the ruler/vernier caliper. Take the average and subtract the initial 8 mm diameter to get the reading. Place the plates back into incubation.
6. Plot a curve with the radial growth in mm (Y-axis) and time (X-axis).

References

Lu, X. H., Hausbeck, M. K., Liu, X. L., & Hao, J. J. (2011b). Wild Type Sensitivity and Mutation Analysis for Resistance Risk to Fluopicolide in Phytophthora capsici. Plant Disease, 95(12), 1535–1541. https://doi.org/10.1094/pdis-05-11-0372

Santos, M., Diánez, F., Sánchez-Montesinos, B., Huertas, V., Moreno-Gavira, A., García, B. E., Garrido-Cárdenas, J. A., & Gea, F. J. (2023c). Biocontrol of Diseases Caused by Phytophthora capsici and P. parasitica in Pepper Plants. Journal of Fungi, 9(3), 360. https://doi.org/10.3390/jof9030360

Wang, Z., Langston, D. B., Csinos, A. S., Gitaitis, R. D., Walcott, R. R., & Ji, P. (2009b). Development of an Improved Isolation Approach and Simple Sequence Repeat Markers To Characterize Phytophthora capsici Populations in Irrigation Ponds in Southern Georgia. Applied and Environmental Microbiology, 75(17), 5467–5473. https://doi.org/10.1128/aem.00620-09

Aim

To observe the morphology of Phytophthora capsici.

Materials Required

1. P. capsici culture
2. Lactophenol Cotton Blue stain
3. Forceps
4. Glass slides
5. Cover slips
6. Microscope

Procedure

1. Place a drop of lactophenol cotton blue on a clean slide.
2. Using a sterile inoculation loop, suspend some P. capsici mycelia in the lactophenol cotton blue on the slide.
3. Cover the slide with a cover slip without any air bubbles.
4. Observe the slide at 10x and 40x with a microscope (we used a Cilika Tab Pro microscope).

Aim

To observe the infection of Phytophthora capsici on Piper nigrum leaves and test our nanoformulation on the same with established controls.

Materials Required

1. Plastic boxes
2. Cotton
3. Autoclaved distilled H₂O
4. Inoculation loop
5. Nichrome wires
6. Toothpicks
7. Spray bottles
8. 1% acetic acid
9. Chitosan Nanoparticles
10. Nanoformulation (siRNA encapsulated in the chitosan nanoparticles)
11. Leaves of Piper nigrum (Black Pepper)
12. Phytophthora capsici subculture, 72 hrs old
13. Forceps
14. 5 mm cork borers
15. Gloves
16. Mask
17. Lab coat
18. Tissues
19. 70% ethanol
20. Parafilm

Procedure

The protocol was adopted from Paul et al. (2019b), and a few modifications were made.

1. Pluck leaves of Piper nigrum (we took the Panniyur 1 variety or any other strongly susceptible species) that are of the same maturity, colour, and size. Ideally, the leaf should be the third from the growing tip. Ensure they are clear of infection, yellowing, and spotting on both sides.
2. Clean the leaves thoroughly under water and gently dry using tissue papers. Wipe with 70% ethanol as well.
3. Airtight plastic boxes are sterilized using 70% ethanol and tissues to prevent contamination. They are then lined with a layer of cotton on the base, keeping the container walls clear for easier visualization.
4. Autoclaved distilled water is poured over the cotton lining until thoroughly soaked. The excess water is drained by tilting the container.
5. Label the boxes as per the experiments with the necessary controls.
6. Place the plastic containers, an inoculation loop, nichrome wires, a few toothpicks, spray bottles filled with the treatment solution, forceps, and 5mm cork borers within the Laminar Air Flow hood, post cleaning with 70% ethanol under ultraviolet radiation for 20 minutes. Ensure GLP is followed; all personnel should wear lab shoes, lab coats, masks, and gloves.
7. After the UV treatment, gently arrange the cleaned leaves in the boxes and press them flat.
8. Using toothpicks, prick marks are to be made uniformly across all leaves at the centre. A 4x4 pattern was followed in all of them.
9. For the control set of leaves, autoclaved distilled water is to be sprayed and allowed to dry completely before inoculation.
10. For the 1% acetic acid control (because the nanoparticles and the nanoformulation are dissolved in 1% acetic acid), the leaves are sprayed with 1% acetic acid, and the leaf is allowed to dry.
11. The same applies to the nanoparticle control and the nanoformulation test. Spray equal volumes onto both the leaves and allow them to dry before introducing the oomycete.
12. A 72 hrs old Phytophthora capsici subculture is to be taken, and 5 mm cork borers (Heat flame before use and let it cool completely) are used to punch out mycelial plugs from the periphery of the subculture in the fungal mat.
13. Carefully, using the inoculation loop (preferably with a new nichrome wire to avoid cross-contamination), and the forceps (heat flame before use and let it cool thoroughly), pick out a mycelial disk and place one at the centre of each leaf where the pricks were made, such that the mycelial side touches the leaf.
14. Parafilm the subculture after taking out the disks and place them back in the dark incubator at 25°C.
15. Heat the inoculation loop thoroughly before and after use and ensure it is cooled down properly to prevent causing any damage to the mycelia. Dispose of the nichrome wire after use.
16. Sterilize the LAF with 70% ethanol post-use.
17. Seal the boxes tightly and place them in a spot with moderate sunlight at room temperature.
18. Lesion readings are to be taken daily, wherein the perpendicular (Y-axis) and the parallel (X-axis) lesion lengths are measured and noted. Take the average of the two values and subtract 5 mm (the diameter of the mycelial plug). Keep a track of the readings until the leaves are completely blackened.
19. Ensure to wear gloves and a mask whilst taking every reading. Sterilize hands with 70% ethanol before and after measuring the lesion growth size. Minimize the duration for which the boxes are kept open.

References

Paul, B. B., Mathew, D., Beena, S., & Shylaja. (2019). Comparative transcriptome analysis reveals the signal proteins and defence genes conferring foot rot (Phytophthora capsici sp. nov.) resistance in black pepper (Piper nigrum L.). Physiological and Molecular Plant Pathology, 108, 101436. https://doi.org/10.1016/j.pmpp.2019.101436

Aim

To determine if varying concentrations of bZIP1 siRNA used for testing show cytotoxic effects on black pepper (Piper nigrum) leaves.

Materials Required

1. Plastic boxes
2. Cotton
3. Autoclaved distilled H₂O
4. Varying concentrations of siRNA in nuclease-free water.
5. Leaves of Piper nigrum (Black Pepper)
6. Gloves
7. Mask
8. Lab coat
9. Tissues
10. 70% ethanol
11. RNase free 10 μL micropipette tips

Procedure

1. Pluck leaves of Piper nigrum (we took the Panniyur 1 variety) are of the same maturity, colour, and size. Ideally, the leaf should be the third from the growing tip. Ensure they are clear of infection, yellowing, and spotting on both sides.
2. Clean the leaves thoroughly under water and gently dry using tissue paper. Wipe with 70% ethanol as well.
3. Airtight plastic boxes are sterilized using 70% ethanol and tissues to prevent contamination. They are then lined with a layer of cotton on the base, keeping the container walls clear for easier visualization.
4. Autoclaved distilled water is poured over the cotton lining until thoroughly soaked. The excess water is drained by tilting the container. Place the containers under ultraviolet light for 20 minutes.
5. After the UV treatment, gently arrange the cleaned leaves in the boxes and press them flat.
6. Using toothpicks, pricks are to be made uniformly across all leaves at the centre. A 4x4 pattern was followed in all of them.
7. Label the boxes as per the siRNA concentrations and necessary controls.
8. In an RNase-free environment, prepare siRNA of varying concentrations (We sourced our siRNA from Juniper Life Sciences ) in RNase-free Eppendorf tubes (Eg: 100 μM, 1 μM, 100 nM, 50 nM, etc).
9. In a laminar airflow chamber, carefully pipette 10 μL of the siRNA solution on the pricked area of each leaf. Ideally, duplicates of each leaf with a particular siRNA concentration should be made.
10. Seal the boxes tightly and place them in a spot with moderate sunlight at room temperature. Observe every 24 hrs.
11. Sterilize the LAF with 70% ethanol post-use.

Aim

To produce and observe zoospores of Phytophthora capsici and test our Nanoformulation on the same with established controls.

Materials Required

1. Hemocytometer
2. Sterilized petri plates
3. Autoclaved distilled H₂O
4. Inoculation loop
5. Nichrome wires
6. Cooling incubator with illumination
7. Glass slides
8. 1% acetic acid
9. Chitosan Nanoparticles
10. Nanoformulation (siRNA encapsulated in the chitosan nanoparticles)
11. Glass coverslip
12. Phytophthora capsici subculture, 72 hrs old
13. Forceps
14. 5 mm cork borers
15. Gloves
16. Mask
17. Lab coat
18. Tissues
19. 70% ethanol
20. Parafilm
21. Micropipettes
22. Micropipette tips
23. 1.5 mL Eppendorf tubes
24. Methylene blue dye

Procedure

1. Place an inoculation loop, nichrome wires, 5 mm cork borers, autoclaved distilled water, and petri plates within the Laminar Air Flow hood, post cleaning with 70% ethanol under ultraviolet radiation for 20 minutes. Ensure GLP is followed; all personnel should wear lab shoes, lab coats, masks, and gloves.
2. 72 hrs old Phytophthora capsici subcultures cultured on carrot agar medium are to be taken, and 5 mm cork borers (Heat flame before use and let it cool completely) are used to punch out several mycelial plugs from the periphery of the subculture in the fungal mat.
3. The zoospore suspension was prepared by carefully using the inoculation loop and the forceps to pick out the mycelial discs and placing 10 of them with the mycelial side facing upwards (the discs must be slightly immersed in water) in each sterile petri plate containing autoclaved sterile distilled water (half-filled with water). Double-parafilm the plates thereafter.
4. The plates were incubated at 25°C under continuous illumination for 48 hours to induce sporangial production.
5. Parafilm the subculture plates after removing the disks and place them back in the dark incubator at 25°C.
6. After 48 hrs, the suspension in the petri plates was cold shocked at 4°C for 30 minutes to facilitate the release of zoospores.
7. Pipette out the zoospore solution and prepare the samples to be tested.
   • Tubes containing the zoospore solution- Negative control (1 mL pipetted out from each plate)
   • Tubes containing zoospore solution + 1% acetic acid solution (500 μL zoospore solution from each plate + 250 μL acetic acid)
   • Tubes containing the zoospore solution + the 0.1% chitosan nanoparticle solution (500 μL zoospore solution from each plate, 250 μL chitosan NP solution)
   • Tubes containing the zoospore solution + the 0.1% siRNA encapsulated chitosan nanoparticle solution (500 μL zoospore solution from each plate, 250 μL siRNA chitosan NP solution- the nanoformulation)
8. Add a few drops of Methylene blue to each sample tube. Pipette out 10 μL of each sample on a clean glass slide and place a clean coverslip on top. Visualize at 10x, 40x, and 100x (we used the Olympus CX-43 upright trinocular phase contrast microscope ).
9. To measure changes in zoospore motility and get the number of zoospores per mL of the solution, pipette the samples carefully on a hemocytometer by carefully placing the tip at the edge of the slide such that the liquid flows below the coverslip by capillary action onto the etched platform.
   To measure changes in mobility, use the hemocytometer and measure the time it takes for a zoospore to move from one side of a square in the grid to the other. To count the number of cells per mL:
   1. Count the number of cells in each square.
   2. Count the number of cells in at least 10 squares (10-20 cells per square).
   3. Repeat and count at least 6 times to obtain minimal variation.

Aim

Synthesis of chitosan nanoparticles using TPP crosslinker by ionic gelation.

Materials Required

1. Chitosan (Molecular Weight ≈ 50,000-190,000 Da)
2. Sodium Tripolyphosphate (TPP)
3. Acetic acid (1% v/v solution)
4. Sterile distilled water
5. Magnetic stirrer
6. 1 N NaOH solution
7. PVDF syringe filters (pore size 0.22 μm)
8. Centrifuge
9. Beakers/flasks
10. Pipettes/measuring cylinders

Procedure

Protocol adopted from (De Carvalho et al., 2019b; Oh et al., 2019b) and optimized for our experimentation.

1. Prepare 1% Acetic acid (1 mL in 100 mL of distilled water). Calibrate the pH meter and measure the pH of the solution (should be 2.6-2.8).
2. Prepare 50 mL of 1 N NaOH solution (weigh 2 g of NaOH pellets and add sterile distilled water up to 50 mL). Simultaneously, turn on the ice machine.
3. Dissolve the low molecular weight chitosan powder at a concentration of 0.1% (w/v) in 1% (v/v) acetic acid (0.04 g in 40 mL of 1% Acetic acid solution).
4. Rinse the magnetic bead in distilled water. Stir the solution in a magnetic stirrer (Eltek MS 205) at maximum stable speed for 30 minutes to ensure complete dissolution.
5. Dissolve sodium tripolyphosphate (TPP) at a concentration of 0.24% (w/v) in sterile distilled water (0.072 g in 30 mL of sterile water).
6. Adjust the pH of the chitosan solution to 4 using 1 N NaOH solution. To do this,
   1. Check the pH of the chitosan solution by taking 5 mL in a Falcon tube.
   2. Determine an estimate of the volume of 1 N NaOH to be added to the solution by adjusting the pH of 1% acetic acid of the same volume.
   3. Accordingly, add the appropriate volume of NaOH to the chitosan solution and check the final pH (using 5 mL of the remaining solution).
7. Sonicate the chitosan solution at 40% amplitude, 10-sec ON/OFF pulse for 5 minutes. Wipe the probe with 70% ethanol before and after use.
8. Filter the chitosan solution through regular filter paper into a beaker to remove any undissolved particles.
9. Syringe filter the TPP solution using a 0.22 μm PVDF filter into a Falcon tube until the volume is 10 mL.
10. Rinse the burette with distilled water and residual filtered TPP solution. Establish the optimal flow rate of roughly 7s between consecutive drops using the same. Once fixed, suspend the magnetic bead in the chitosan solution and begin stirring.
11. Load 10 mL of the TPP solution onto the burette and add drop by drop to 30 mL of the chitosan solution. Ensure the drops fall at the periphery of the vortex.
12. Once 10 mL of the TPP solution has been added, stir the nanoparticle solution for another 20 minutes. Transfer the solution to a 50 mL Falcon tube.
13. Sonicate the nanoparticle solution at 40% amplitude, 10-sec ON/OFF pulse for 8 minutes.
14. Centrifuge (we used the Eppendorf centrifuge 5804 R) the nanoparticle solution at 1000 rpm for 5 minutes at room temperature. Carefully place the glass pipette on the side of the Falcon tube that faced inwards during centrifugation. Ensure the orientation of the falcon tube is maintained throughout the process. Decant up to 20 mL of the solution into a Falcon tube. This serves as the nanoparticle solution.
15. Characterize the samples using a particle size analyzer and scanning electron microscope (we used the Horiba Scientific nanopartica nano particle analyzer SZ-100 and VO MA18 with Oxford EDS(X-act) scanning electron microscope). Store this solution at 4°C.

References

De Carvalho, F. G., Magalhães, T. C., Teixeira, N. M., Gondim, B. L. C., Carlo, H. L., Santos, R. L. D., De Oliveira, A. R., & Denadai, Â. M. L. (2019). Synthesis and characterization of TPP/chitosan nanoparticles: Colloidal mechanism of reaction and antifungal effect on C. albicans biofilm formation. Materials Science and Engineering C, 104, 109885. https://doi.org/10.1016/j.msec.2019.109885

Oh, J.-W., Chun, S. C., & Chandrasekaran, M. (2019). Preparation and In Vitro Characterization of Chitosan Nanoparticles and Their Broad-Spectrum Antifungal Action Compared to Antibacterial Activities against Phytopathogens of Tomato. Agronomy, 9(1), 21. https://doi.org/10.3390/agronomy9010021

Aim

Encapsulation of siRNA in chitosan nanoparticles using the ionic gelation method.

Materials Required

1. Chitosan (Molecular Weight ≈ 50,000-190,000 Da)
2. siRNA of desired volume and concentration
3. Sodium Tripolyphosphate (TPP)
4. Acetic acid (1% v/v solution)
5. RNase-free water
6. Magnetic stirrer
7. PVDF syringe filters (pore size 0.22 μm)
8. Centrifuge
9. Beakers/flasks
10. Pipettes/measuring cylinders

Procedure

Protocol adopted from (Jarudilokkul et al., 2011b; Katas & Alpar, 2006d; Raja et al., 2015c).

1. Ensure all glassware, plasticware, and reagents prepared are RNase-free.
2. Prepare 1% Acetic acid (1 mL in 99 mL of RNase-free water). Calibrate the pH meter and measure the pH of the solution.
3. Dissolve the low molecular weight chitosan powder at a concentration of 0.1% (w/v) in 1% (v/v) acetic acid (0.04 g in 40 mL of 1% Acetic acid solution).
4. Rinse the magnetic bead in RNase-free water. Stir the solution in a magnetic stirrer (Eltek MS 205) at maximum stable speed for 30 minutes to ensure complete dissolution.
5. Dissolve sodium tripolyphosphate (TPP) at a concentration of 0.24% (w/v) in RNase-free water (0.072 g in 30 mL of sterile water).
6. Sonicate the chitosan solution at 40% amplitude, 10-sec ON/OFF pulse for 5 minutes. Wipe the probe with 70% ethanol before and after use.
7. Filter the chitosan solution through normal filter paper into a beaker to remove undissolved particles.
8. Syringe filter the TPP solution using a 0.22 μm PVDF filter into a Falcon tube until the volume is 10 mL.
9. Add the desired siRNA concentration resuspended in nuclease-free water to the TPP solution in a laminar air flow chamber. We added 15 μL of 100 μM siRNA to 10 mL of TPP (giving a 1:666 ratio of siRNA to TPP). Ensure RNase-free tips are used and all surfaces are RNase-free.
10. Rinse the burette with RNase-free water and residual filtered TPP solution. Establish the optimal flow rate of roughly 7s between consecutive drops using the same. Once optimized, suspend the magnetic bead in the chitosan solution and begin stirring.
11. Load 10 mL of the siRNA-TPP solution onto the burette and add drop by drop to 30 mL of the chitosan solution. Ensure the drops fall at the periphery of the vortex.
12. Once 10 mL of the siRNA-TPP solution has been added, stir the nanoparticle solution for another 20 minutes. Transfer the solution to a 50 mL Falcon tube.
13. Sonicate the encapsulated siRNA solution at 40% amplitude, 10-sec ON/OFF pulse for 10 minutes.
14. Centrifuge the encapsulated siRNA solution at 1000 rpm for 5 minutes at room temperature. Ensure the orientation of the falcon tube is maintained throughout the process. Carefully place the glass pipette on the side of the Falcon tube that faced inwards during centrifugation. Decant up to 20 mL of the solution into a Falcon tube. Store at 4°C.

References

Jarudilokkul, S., Tongthammachat, A., & Boonamnuayvittaya, V. (2011). Preparation of chitosan nanoparticles for encapsulation and release of protein. Korean Journal of Chemical Engineering, 28(5), 1247–1251. https://doi.org/10.1007/s11814-010-0485-z

Katas, H., & Alpar, H. O. (2006b). Development and characterisation of chitosan nanoparticles for siRNA delivery. Journal of Controlled Release, 115(2), 216–225. https://doi.org/10.1016/j.jconrel.2006.07.021

Raja, M. A. G., Katas, H., & Wen, T. J. (2015b). Stability, Intracellular Delivery, and Release of siRNA from Chitosan Nanoparticles Using Different Cross-Linkers. PLoS ONE, 10(6), e0128963. https://doi.org/10.1371/journal.pone.0128963

Aim

To calculate the entrapment efficiency of siRNA in chitosan nanoparticles using a UV-vis spectrophotometer.

Materials Required

1. Centrifuge
2. UV-vis spectrophotometer
3. RNase-free water
4. Nanocuvette
5. Encapsulated siRNA in chitosan nanoparticle (suspended in 1% acetic acid)
6. 70% ethanol

Procedure

Protocol adopted from (Katas & Alpar, 2006e; Raja et al., 2015d).

1. Centrifuge the siRNA-loaded chitosan nanoparticle sample at 11,000 rpm and 4°C for 37 minutes.
2. Carefully remove the supernatant from the siRNA-loaded CS-TPP nanoparticles. Clean the nanocuvette using 70% ethanol.
3. Load 2 μL of 1% acetic acid as a blank in the nanocuvette and measure absorbance at 260 nm using a UV-vis spectrophotometer.
4. Load 2 μL of the supernatant into the nanocuvette and measure absorbance at 260 nm using a UV-vis spectrophotometer (we use the Eppendorf BioSpectrometer). Ensure an RNase-free environment is maintained throughout the experiment, and clean the nanocuvette with 70% ethanol between uses.
5. Calculate the entrapment efficiency using the formula:
   

   Entrapment Efficiency (%) = C_sample − C_supernatant C_sample × 100

   
   C_sample is the concentration of siRNA added, and C_supernatant is the siRNA concentration in the supernatant.

References

Katas, H., & Alpar, H. O. (2006). Development and characterisation of chitosan nanoparticles for siRNA delivery. Journal of Controlled Release, 115(2), 216–225. https://doi.org/10.1016/j.jconrel.2006.07.021

Raja, M. A. G., Katas, H., & Wen, T. J. (2015b). Stability, Intracellular Delivery, and Release of siRNA from Chitosan Nanoparticles Using Different Cross-Linkers. PLoS ONE, 10(6), e0128963. https://doi.org/10.1371/journal.pone.0128963

Aim

To determine if encapsulation of the siRNA in chitosan nanoparticles has been successfully achieved by visualizing on a gel.

Materials Required

1. Encapsulated siRNA chitosan nanoparticle solution
2. Naked siRNA
3. Chitosan nanoparticle solution
4. RNase
5. Low EEO Agarose
6. 1x TBE Buffer
7. DNA Ladder
8. RNA Loading Dye
9. SyberSAFE
10. RNase-free tips and PCR tubes
11. Autoclaved Milli-Q water
12. Microwave oven
13. Gel electrophoresis comb
14. Gel Casting Tray
15. Conical flask
16. Graduated cylinder
17. UV transilluminator
18. Gel electrophoresis unit

Prepare the buffer and gel with Autoclaved Milli-Q water. Store all reagents (except siRNA, chitosan nanoparticle solution, encapsulated siRNA chitosan nanoparticle solution, RNA loading dye) at room temperature.

Procedure

Protocol adopted from (Helling et al., 1974; Katas & Alpar, 2006f; Rio et al., 2010; Scott et al., 2003).

1. Make 1x TBE Buffer by diluting the 50x TBE buffer in autoclaved Milli-Q water. For 1 L of 1x TBE buffer, add 20 mL 50x TBE buffer to 980 mL of autoclaved Milli-Q water.
2. Prepare 2% agarose gel (35 mL): Weigh 0.7 g Low EEO agarose powder, and add 35 mL of 1x TBE buffer. After that, boil the agarose mixture in a microwave oven to melt it. Cool the solution to approximately 50–60 °C, add 1.16 μL of SybrSAFE, and mix well. Pour the gel into the gel casting tray with the comb attached. Using an RNase-free pipette tip, gently push aside any floating bubbles to the top.
3. Once the gel is solidified, place it in the 1x TBE buffer poured into the gel electrophoresis unit. Carefully remove the comb, ensuring no tears in the wells.
4. In an aseptic and RNase-free environment, prepare the samples for loading. Pipette 10 μL of each sample in RNase-free PCR tubes: chitosan nanoparticle solution, encapsulated siRNA in CSNP, naked siRNA, and siRNA in CSNP treated with RNase (2 μL of 0.5 mg/mL RNase, incubated for 5 minutes at 56 °C). A 1:6 dilution of loading dye was added to each well (2 μL in each well).
5. Spin down the samples for a few seconds and load them into the gel. Load 10 μL RNA ladder as well.
6. Carry out the electrophoresis at a voltage of 55 V for 2 hrs, checking the gel in intervals.
7. Visualize the siRNA bands under a UV transilluminator at a wavelength of 365 nm.

References

Helling, R. B., Goodman, H. M., & Boyer, H. W. (1974). Analysis of endonuclease R· Eco RI fragments of DNA from lambdoid bacteriophages and other viruses by agarose-gel electrophoresis. Journal of virology, 14(5), 1235-1244. https://doi.org/10.1128/jvi.14.5.1235-1244.1974

Katas, H., & Alpar, H. O. (2006c). Development and characterisation of chitosan nanoparticles for siRNA delivery. Journal of Controlled Release, 115(2), 216–225. https://doi.org/10.1016/j.jconrel.2006.07.021

Rio, D. C., Ares, M., Hannon, G. J., & Nilsen, T. W. (2010). Nondenaturing agarose gel electrophoresis of RNA. Cold Spring Harbor Protocols, 2010(6), pdb-prot5445. https://cshprotocols.cshlp.org/content/2010/6/pdb.prot5445.short

Scott, V., Clark, A. R., & Docherty, K. (1994). The gel retardation assay. Protocols for gene analysis, 339-347. https://link.springer.com/protocol/10.1385/0-89603-258-2:339

Aim

To visualize the internalization of the 6-FAM-tagged siRNA within Phytophthora capsici zoospores.

Materials Required

1. Phytophthora capsici zoospores in autoclaved distilled water.
2. 6-FAM tagged siRNA
3. RNase-free Eppendorf tubes
4. RNase-free micropipette tips
5. Micropipette
6. Glass slides
7. Methylene blue
8. Hemocytometer
9. siRNA encapsulated in CSNP
10. 100 ng/mL siRNA
11. Nuclease-free water
12. Fluorescence microscope

Procedure

Protocol adopted from (Cheng et al., 2022c; M. Wang et al., 2016b).

1. Cold shock the 48 hrs incubated zoospore solution for 30 minutes at 4°C. Pipette 250 μL of the solution into a 1.5 mL Eppendorf tube and add 1 or 2 drops of Methylene blue dye. Calculate the concentration of the zoospores per mL of solution using a hemocytometer. Dilute the zoospore suspension to the desired concentration.
2. Prepare the zoospore samples for testing. Add 4 μL of 100 ng/mL siRNA to 6 μL of zoospore solution of 10⁵ spores per mL to get a final siRNA concentration of 40 ng/mL. Prepare zoospore samples treated with siRNA encapsulated in CSNP. Use 10 μL of 10⁵ spores per mL as a negative control.
3. Incubate the samples in the dark for 0.5 hrs to 4 hrs at room temperature, giving the siRNA enough time to be taken up by the zoospores.
4. Pipette the samples onto clean glass slides and place a cover slip without any air bubbles.
5. Visualize the samples using a fluorescence microscope at 10x, 40x, and 100x magnification at 488 nm wavelength (6-FAM has an excitation maximum that closely matches the 488 nm spectral line of the argon-ion laser) of the fluorescence microscope (we used an Olympus IX 73 Fluorescent Microscope).

References

Cheng, W., Lin, M., Chu, M., Xiang, G., Guo, J., Jiang, Y., Guan, D., & He, S. (2022b). RNAi-Based Gene Silencing of RXLR Effectors Protects Plants Against the Oomycete Pathogen Phytophthora capsici. Molecular Plant-Microbe Interactions, 35(6), 440–449. https://doi.org/10.1094/mpmi-12-21-0295-r

Wang, M., Weiberg, A., Lin, F., Thomma, B. P. H. J., Huang, H., & Jin, H. (2016). Bidirectional cross-kingdom RNAi and fungal uptake of external RNAs confer plant protection. Nature Plants, 2(10). https://doi.org/10.1038/nplants.2016.151