Experiment training
We learnt to culture the 293t cells.
We learnt to extract plasmids and transfect it into 293t cells.
We learnt to collect exosomes.
We learnt to extract RNA from exosomes and cells.
We learnt to do RT-PCR and qPCR.
siRNA screening (Failed)
14th: We extracted plasmids and cultured the 293t cells.
15th: We transfected plasmids into 293t cells.
16th: We collected cells and preserved it in Trizol.
17th: We extracted RNA, then did RT-PCR with the RNA.
18th: We did qPCR with the cDNA we got on 17th April.
21th: We reexamined the concentration of RNA, and redid qPCR.
siRNA screening (Failed)
27th: We cultured the 293t cells in a 12-well plate.
28th: We transfected 293t cells.
29th: We collected cells and extracted RNA from it.
30th: We did RT-qPCR with the RNA we extracted on 29th April.
1st: We did qPCR with the cDNA we got on 30th April.
siRNA screening (Failed), examination whether siRNA are capsulated into exosomes (Success)
8th: We extracted plasmids of 3 kinds of siRNA.
9th: We cultured 293t cells in eight D10 dishes and a 12-well plate.
10th: We extracted 2 kinds of over-expression plasmids and examined the concentration of it. Then we transfect these plasmids into 293t cells respectively.
11th: We collected cells and exosomes from 8 D10 dish, and cells from 12-well plate.
12th: We extracted RNA from cells and exosomes.
13th: We did RT-PCR and qPCR.
siRNA screening (Failed)
15th: We cultured the 293t cells in a 12-well plate.
16th: We transfected 293t cells.
17th: We collected cells and extracted RNA from then.
18th: We did RT-PCR and qPCR.
siRNA screening (Failed)
27th: We cultured the 293t cells in 2 12-well plates.
28th: We transfected 293t cells, with lipofectamine 2000.
29th: We collected cells and extracted RNA from them.
30th: We continued to extract RNA, and examine the concentration of total RNA. Then we did RT-PCR.
31st : We did qPCR.
siRNA screening (failed)
8th: We cultured Hep G2 cells in 2 12-well plates for 24-hour cell collection and 48-hour cell collection.
9th: We transfected cells with empty Lipo, HbsAg plasmids, HbsAg plasmids and 467 siRNA plasmids, HbsAg plasmids and 516 siRNA plasmids, respectively.
10th: We collected cells 24 hours after transfection, and preserved them in Trizol.
11th: We collected 48 hours after transfection, and then extracted RNA from 2 groups of cells (24 hours and 48 hours).
12th: We did RT-PCR and qPCR with all RNA samples.
13th: We analyzed the data.
siRNA screening (failed)
8th: We cultured Hep G2 cells in 2 12-well plates for 24-hour cell collection and 48-hour cell collection.
9th: We transfected cells with empty Lipo, HbsAg plasmids, HbsAg plasmids and 467 siRNA plasmids, HbsAg plasmids and 516 siRNA plasmids, respectively.
10th: We collected cells 24 hours after transfection, and preserved them in Trizol.
11th: We collected 48 hours after transfection, and then extracted RNA from 2 groups of cells (24 hours and 48 hours).
12th: We did RT-PCR and qPCR with all RNA samples.
13th: We analyzed the data.
Absolute quantification of exosomes (failed)
5th: We cultured 293t cells.
6th: Cells we cultured on 5th July were contaminated by bacteria.
7th: We subcultured another cell line of 293t cells.
9th: We cultured 293t cells in 6 flasks.
10th:We transferred 293T cells with plasmids.
11th: We collected exosomes 24 hours after transfection.
12th: A part of cells died, failed to collect 48-hour exosomes.
13th: We examined protein concentration of 24-hour exosomes, started to extract RNA from 24h-cells and 24h-exosomes and preserved the rough RNA extract solution with isopropyl alcohol at 4℃ overnight.
14th：We continued finish RNA extraction, then did RT-PCR and qPCR.
15th: We analyzed data.
Luciferase Assay
11th: Construction of plasmids: obtain vector and segment.
12th: We combined vector and segment with T4 ligase, then transformed the recombined plasmids into E.coli DH5α competent cells and spread them on solid LB culture plates by streak plate method.
13th: No single bacterial colony was found.
14th: No single bacterial colony was found again, redid previous steps(obtain the target segments, combined vector and segment, and then transferred it into E.coli )
15th: Single bacterial colonies were found. We picked single colonies and transferred them into liquid LB culture medium with ampicillin, and shaken overnight at 37℃.
16th: The transformed E.coli cells failed to reproduce in LB culture medium.
Others
5th: We thawed 293t cells.
6th: We subcultured HepG2 cells.
7th: E.coli cells containing HBSag overexpressed plasmids were transferred respectively into LB culture medium with ampicillin, and shaken overnight at 37℃.
8th: We extracted HBSag overexpressed plasmids from E.coli cells.
11th: We thawed 293t cells and subcultured HepG2 cells. E.coli cells containing GFP plasmids were transferred into LB culture medium with ampicillin, and shaken overnight at 37℃.
12th: We extracted GFP plasmids from E.coli cells.
14th: We thawed HepG2 cells and 293T cells.
15th: We thawed 293T cells.
Others
16th: We subcultured 293T cells. E.coli DH5α competent cells containing GFP plasmids and RVG plasmids were transferred respectively into LB culture medium with ampicillin, and shaken overnight at 37℃.
17th: We found that wrong antibiotics were added into the culture medium of RVG plasmid-containing E.coli cells (it ought to be kanamycin). So we did transferred E.coli cells containing RVG plasmids into LB culture medium with kanamycin), and shaken overnight at 37℃.
18th: We preserved the RVG and GFP strains at -80℃ and extracted plasmids from the culture medium (RVG,GFP, 1L medium). And cryopreserved 293T cells.
Absolute quantification of exosomes (succeeded)
18th: We cultured 293t cells.
19th: We transfected 293T cells with 467 and 516 plasmids(lipo*2,467*2,516*2).
20th: We changed the transfection medium with culture medium and then collected culture medium containing exosomes 24 hours after transection (pre-centrifuge).
21th: We collected culture medium containing exosomes 48 hours after transfection (pre-centrifuge) and soaked. centrifuge tubes which then be used in ultracentrifugation overnight in DEPC solution(1:1000).
22th: We separated exosomes by ultracentrifugation(110000g).
23th: We examined protein concentration of exosomes collected by ultracentrifugation, and then extracted RNA from these exosomes and cells which we used to produce exosomes and preserved the RNA extracts in isopropyl alcohol over night.
24th：We continued to extract RNA, examined total RNA concentration of exosomes and cells, then RT-PCR RNA 467 and 516.
25th: Q-PCR the cDNA of RNA 467 and 516.
26th: We analyzed data and found that the standard curve cannot be used.
27th: We redid the Q-PCR and analyzed data and this time we succeeded
Examination whether RVG-lamp2b exosomes will target to dendritic cells or not (failed)
18th: We cultured 293t cells.
19th: We transfected 9 flasks of 293T cells with RVG plasmids(lipo*3,RVG*3,non-related plasmids*3).
20th: We changed the 19th transfection medium with cell culture medium 6 hours after transfection and transfected another 9 flasks of 293T cells as we did with the former 9 flasks.
21th: We collected 48-hour cell culture medium transfected on 19th July and pre-centrifuged to remove cell debris and organelles. We soaked centrifuge tubes which then be used in ultracentrifugation overnight in DEPC solution(1:1000).
22th: We collected 48-hour culture transfected on 20th July and pre-centrifuged to remove cell debris and organelles. Meanwhile, we watched green fluorescent in cells. Then we separated exosomes by ultracentrifugation (110000g).
23th: We examined protein concentration of exosomes collected on 22th July, diluted exosome solution from 500μL to 1000μL and filtered the exosome solution then injected exosome solution into the C57 mice. We took 3μL exosomes solution to extract RNA, and preserved RNA extracts in isopropyl alcohol over night.
24th：We continued to extract RNA, and examined total RNA concentration of exosomes and cells which we used to produce these exosomes, then RT –PCR.
25th: We injected exosome solution into the mice for the second time. Q-PCR the cDNA got from RT-PCR on 24th July.
26th: We analyzed data and found the standard curve cannot be used.
27th: We anatomized C57 mice and collected the brain, heart, liver, spleen, lung, kidney and blood of them and redid RT-PCR and Q-PCR with the RNA extracted on 24th July and analyzed data.
28th: We extracted RNA from brain and serum collected on 27th July
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