1. Transfection of chassis and isolation of exosomes
HEK293 cells were seeded in 225-cm2 flasks (Corning). When the cells reached approximately 70-80% confluence, they were co-transfected with plasmids we used using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. The cell culture medium was then harvested 48 h after transfection, and the exosomes loaded with siRNA were harvested from the medium using an exosome isolation kit (Invitrogen) according to the manufacturer’s instructions. The resulting pellet was then resuspended in PBS.
2. Transmission electron microscopy
For transmission electron microscopy analysis, the exosome samples were prepared as described above. Briefly, the exosome pellet was placed in a droplet of 2.5% glutaraldehyde in PBS buffer and fixed overnight at 4 °C. The exosome samples were rinsed 3 times in PBS for 10 min each and then fixed in 1% osmium tetroxide for 60 min at room temperature. Then, the samples were embedded in 10% gelatine, fixed in glutaraldehyde at 4 °C and cut into small blocks (less than 1 mm3). The samples were dehydrated in increasing concentrations of alcohol. Then, the samples were placed in propylene oxide and infiltrated with increasing concentrations of Quetol-812 epoxy resin mixed with propylene oxide for 3 h per step. Finally, the samples were embedded in pure, fresh Quetol-812 epoxy resin and polymerised at 35 °C for 12 h, 45 °C for 12 h, and 60 °C for 24 h. Ultrathin sections were cut using a Leica UC6 ultra-microtome and stained with uranyl acetate for 10 min and lead citrate for 5 min at room temperature. The samples were then observed with a transmission electron microscope (JEM-1010) at a voltage of 80 kV.
3. Incubating cells with exosomes
Exosomes (100 μg) loaded with siRNAs were incubated with LLC cells (106 cells). After 24h incubation, the recipient cells were collected for total RNA extraction and subsequent quantitative RT-PCR analysis of siRNA and mRNA, and for total protein isolation and subsequent western blotting analysis of protein.
4. Mammalian Adherent Cell Total RNA isolation using TRIzol reagent
Reagents:
TRIzol(Life Technologies，15596-026);
DEPC-treated water(Life Technologies，4387937);
75% DEPC ethanol;
PBS;
Chloroform;
Isopropanol;
Protocol:
(1) Discard culture media from the culture dish;
(2) Wash the cell with 2ml PBS once to make sure culture media is completely removed;
(3) Add 1ml TRIzol reagent every 1 x 106 cells;
(4) Pipette several times to permit complete dissociation of the cell. Transfer the liquid to a
clean 1.5ml tube;
(5) Add 0.2 mL of chloroform per 1 mL of TRIzol Reagent used for homogenization. Vortex vigorously
to mix thoroughly. Place 2-3min at room temperature;
(6) Centrifuge at 16000 x g at 2-8℃ for 15 minutes;
(7) Remove the aqueous phase of the sample by angling the tube at 45° and pipetting the solution out
to a clean 1.5ml tube.
(8) Add 0.5ml of 100% isopropanol to the aqueous phase, per 1ml of TRIzol Reagent;
(9) Incubation at -20℃ for at least one hour;
(10) Centrifuge at 16000 x g at 2-8℃ for 20 minutes;
(11) Remove the supernatant from the tube, leaving only the RNA pellet;
(12) Wash the pellet, with 1ml of 75% ethanol per 1ml of TRIzol Reagent;
(13) Vortex the sample briefly, then centrifuge the tube at at 16000 x g at 2-8℃ for 10
minutes;
(14) Vacuum or air dry the RNA pellet for 5-10 minutes.
(15) Resuspend the RNA pellet in RNase-free water;
(16) Store at -20℃ for a short time or store at -70℃for long time preservation.
5. microRNA Detection by q-RT-PCR
Reagents:
AMV Reverse Transcriptase XL for RT-PCR (TaKaRa，2630A) ;
dNTP mixture (TaKaRa，4030);
Taq (TaKaRa，DR100A);
miRNA probe;
U6 probe;
Protocol:
(1) Reverse Transcription
Oligo d(T) 18 can be used as primers as mRNA 3’ prime end with a poly(A) tail.
(a) Components: total volume 10μL
5× AMV buffer 2μL AMV 0.5μL dNTPs mixture(10mmol) 1μL Oligo d(T) 18(50μM)(10×) 0.5μL RRI(40U/μl)
0.25μl RNA 2μg DEPC up to 10μL
(b) Cycling Conditions:
Step 1: 42℃, 60min
Step 2: 85℃, 5min
Step 3: 4℃, infinite
(2) qPCR
(a) Components: total volume 20μL
10×buffer 2μL dNTPs mixture (10mmol) 0.4μL MgCl 2 1.2μL Taq 0.2μL Sense primer (10mM) 0.5μL
Antisense primer (10mM) 0.5μL ddH O 13.1μL cDNA 1μL
(b) Cycling Conditions:
Step 1: 95℃, 5min
Step 2: 95℃, 30s
Step 3: 95℃, 30s
Step 4: 95℃, 30s (fluorescence detection)
Step2-Step4, 40 cycles (variable, can be up to 45 cycles)
(c) Data Analysis: The Comparative Ct Method (ΔΔCT Method)
CT---cycles when the reaction reach the threshold, the relative expression level of each miRNA
compares to endogenous Control can be described as 2-ΔCT, (ΔCT= CT sample- CT endogenous control).
U6, a housekeeping gene, is usually used as endogenous Control for miRNA.
6. Mammalian cell total protein isolation by RIPA lysi
Reagents:
RIPA lysis (medium) (Beyotime, P0013C);
SDS-PAGE Loading Buffer (5 x) (Beyotime，P0015F);
Protocol:
(1) Discard the culture media;
(2) Add 80 μl RIPA, scrape off the adherent cells;
(3) Transfer the cells to a clean 1.5ml tube;
(4) Incubation on ice for 30 min;
(5) Centrifuge at 12000rpm for 15min at 4℃;
(6) Transfer the supernatant, add 1 μL SDS-PAGE loading buffer every 4μL supernatant;
(7) Boil to denature the protein at 98℃ for 5min, store at -80℃;
7. Western Blot
(1) Prepare Solutions
(2) Gel Electrophoresis
(a) Load protein and molecular weight marker
(b) Add running buffer
(c) Electrify: set the voltage at 80V before the sample reach the dividing line between stacking gel
and resolving gel, switch to 120V until the blue stain reach the buttom line;
(3) Blotting
I. Transfer
(a) Carefully cut the gel
(b) Assemble the transfer cassette
(c) Install the cassette and electrify to transfer the protein to PVDF
II. Chemiluminescene
(a) Blocking: 5% skimmed milk incubate for 1h
(b) Incubate with diluted primary antibody
(c) Wash membrane: TBST 15min *4
(d) Incubate with diluted secondary antibody
(e) Wash membrane: TBST 15min *4
(f) Add Chemiluminescene substrate
(g) Exposure
8. Transfection of Mammalian Adherent Cells
Reagents:
Lipofectamine 2000 Transfection Reagent(Life Technologies，11668019);
DMEM;
Opti MEM;
RPMI 1640;
Fetal Bovine Serum;
Protocol:
(1) When the mammalian cells reached approximately 70%-80% confluence, they were
transfected with the labelled siRNA;
(2) Transfection components;
(3) Sample, preparation:
(a) Dilute DNA/RNA in serum-free Opti-MEM or reduced serum culture (1:4), mix by pipette
gently;
(b) Dilute Lipofectamine 2000 in serum-free Opti-MEM or reduced serum culture (1:4), incubate at
room temperature for 5 min;
(c) Mix the diluted DNA/RNA and the diluted Lipofectamine 2000, incubate for 5min;
(4) Discard the culture media;
(5) Wash the cell with 2ml PBS once to make sure culture media is completely removed;
(6) Change the culture media to serum-free Opti-MEM or reduced serum (1%-2%)DMEM/ RPMI 1640;
(7)Add DNA/RNA and Lipofectamine 2000 mixture to culture media, mix thoroughly by gently rotating
the culture vessel，incubate at 37°C in 5% CO2.
(8) Optional: Change to complete culture media 4-6h after transfection.