siRNA transfection protocol for hBMSCs for RNA analysis, Western blots and
Chemotaxis
(MDA231 cells used as contol)
Day 0 (day before transfection):
Seed cells late in the afternoon, and then transfect them the next morning
Seed cells in normal growth media (hBMSCs in 15% FBS aMEM, DMA231 cells in 10%
FBS DMEM)
- for RNA (to be collected 48 hrs after transfection):
hBMSCs --1*105cells/6-well
MDA231 cells--3*105cells/6-well
- for protein (to be collected 72 hrs after transfection):
hBMSCs –3*105cells/60mm dish
MDA231 cells--8*105cells/60mm dish
- for Chemotaxis (to be collected 72 hrs after transfection):
hBMSCs --1*105cells/6-well
MDA231 cells--3*105cells/6-well
Day1 (Transfection, 80% confluent)
1. For each RNA duplex sequence: make 20uM stock using Rnase-free water
EGFR siRNA from IDT DsiRNA TriFECTa kit which including 3 different
sequences for target gene, so I also combined them and made a mixture of them.)
2. Make A and B by mixing siRNA and serum-free media (Opti-MEM, Invitrogen),
and transfection reagent (Lipofectmine2000, Invitrogen) and serum-free media as
follows for each 6-well plate (total 2mL of media per well) or 60mm dish(total
4mL of medis per dish):
A
20uM siRNA
Serum-free media
Transfection reagent
Serum-free media
B
3.
4.
5.
6.
For final conc. of 20nM
2 ul
50ul
7ul
200ul
Incubate Mixture A and B for 5 min at RT,
Mix A and B, then incubate at RT for 20min.
Meanwhile, change to fresh growth media for cells
After incubation, add 250ul of A + B mixture dropwise per 6-well
Day 3: Collect for RNA analysis
Day 4: Chemotaxis analysis and Collect for Western blots