Difference between revisions of "Team:NJU-China/Notebook/Protocol"

HEK293 cells were seeded in 225-cm2 flasks (Corning). When the cells reached approximately 70-80% confluence, they were co-transfected with plasmids encoding Lamp2b-iRGD and KRAS siRNA 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 MOR 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.

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.

Exosomes (100 μg) loaded with KRAS siRNAs were incubated with A549 cells (106 cells). After 24 h incubation, the recipient cells were collected for total RNA extraction and subsequent quantitative RT-PCR analysis of KRAS siRNA and KRAS mRNA, and for total protein isolation and subsequent western blotting analysis of KRAS protein.

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.

Reagents:
AMV Reverse Transcriptase XL for RT-PCR (TaKaRa，2630A)
dNTP mixture (TaKaRa，4030);
Taq (TaKaRa，DR100A);
miRNA probe;
GAPDH 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
MgCl2	1.2μL
Taq	0.2μL
Sense primer (10mM)	0.5μL
Antisense primer (10mM)	0.5μL
ddHO	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). GAPDH, a housekeeping gene, is usually used as endogenous Control for mRNA.

Reagents: