Transfection of S. mansoni schistosomula with siRNA/PEI nanoparticle complexes
April 7-10, 2014
Biomedical Research Institute, Rockville, MD
Day 1
9:00-9:30 A.M.
9:30-10:30 A.M.
10:30-10:45 A.M.
10:45-11:45 A.M.
Lecture Dr. Matt Tucker (Biomedical Research Institute, Rockville, MD.)
‘Welcome and introduction’
Lecture Dr. Paul J. Brindley (The George Washington University, Washington, D.C.)
‘Transgenesis in schistosomes’
Break
Lab.
part I
- Brief lab. Procedure for S. mansoni Cathepsin B (Cath B). gene suppression by siRNA,
(Dr. Wannaporn Ittiprasert, BRI)
- siRNA sysnthesis, part 1 (Wannaporn Ittiprasert and Andre´ Miller)
11:45 A.M.-1:00 P.M.
1:00-1:30 P.M.
1:30-2:30 P.M.
2:45-3:00 P.M.
3:00-3:30 P.M.
Lunch
Lab.
9:00-9:30 A.M.
9:30-10:30 A.M.
Lab.
- siRNA sysnthesis, part 4 (WI & AM)
Lecture Dr. Gabriel Rinaldi (George Washington University, Washington, D.C.)
‘RNAi in flatworms; history, approaches and perspectives’
10:30-10:45 A.M.
10:45-11:45 A.M.
Break
Lab.
- Schistosomula collection and in vitro culture (WI & AM)
11:45 A.M.-1:00 P.M.
1:00-2:45 P.M.
Lunch
Lab.
- siRNA purification (WI & AM)
2:45-3:00 P.M.
3:00-3:30 P.M.
Break
Lab.
- siRNA quantification (WI & AM)
9:00-10:15 A.M.
Lecture Dr. Patrick Skelly (Tufts University, North Grafton, MA.)
10:15-10:30 A.M.
10:30-11.30 A.M.
Break
Lab.
Break
Lab.
- siRNA sysnthesis, part 2 (WI & AM)
- Brief lab. Procedure for S. mansoni Cath B gene suppression by siRNA, part II (WI)
- siRNA sysnthesis, part 3 (WI & AM)
Day 2
Day 3
‘RNAi in Schistosomes’
- siRNA-PEI complexation (WI & AM)
- siRNA/PEI nanoparticle transfection (WI & AM)
* This experiment includes Cy3 labeled- siRNA negative control and plasmid
DNA containing GFP gene for further success transfection test
11:30 A.M.-1:00 P.M.
1:00-2:30 P.M
Lunch
Lab.
2:30-2:45 P.M.
2:45-3:30 P.M.
Break
Lab.
- Total RNA and DNA qualification and quantification (WI & AM)
9:00-10.15 A.M.
Lab.
- Semi-quantitative RT-PCR for Cath B and PCR for GFP (WI & AM)
10:15-10:30 A.M.
10:30-11:10 A.M.
11:45 A.M.-1:00 P.M.
1:00-2:30 P.M.
Break
Lecture Dr. Dierdre Joy (DMID, NIAID, National Institutes of Health, MD.)
‘NIH Grants and Research Resources’
Lecture Dr. Robert Molestina (BEI Resources, ATCC, Manassas, VA.)
‘BEI Resources: A Biological Resource Center for Parasitologists’
Lunch
Lab.
- Agarose gel electrophoresis (WI & AM)
2:30-2:45 P.M.
2.45-3.30 P.M.
Break
Lab.
- Total RNA extraction (WI) from siRNA transfected schistosomula
- Genomic DNA extraction (AM) from pNL-GFP transfected schistosomula
Day 4
11:10-11:45 A.M.
- Fluorescent microscopy examination for transfected parasite (Dr. Matt Tucker)
- Agarose gel imaging (AM)
- Conclusion