3.1.1 Preparation
of Culture Medium
and Fibronectin-Coated
Plates
1. Prepare complete culture medium to reach a final concentration of 10% FBS, 1% sodium pyruvate solution, and 1%
penicillin-streptomycin solution in DMEM.
2. Coat the 150 mm dishes with fibronectin: Rinse the 150 mm
dishes with 10 mL PBS. Add 10 mL of the fibronectin working
solution to each dish and incubate at 37
C for 20 min. After
incubation, rinse the dish with 10 mL PBS.
3.1.2 Preparation
of “Mix” Control Samples
As a control for the transfer of RNA between cells in co-culture, we
strongly recommend having a negative control of the two cell types
mixed together (“mix”) and immediately sorted (see Subheading
3.2). Proceed as follows:
1. Culture the different cell types in fibronectin-coated dishes,
prepared as described in Subheading 3.1.1.
2. Harvest the separately cultured MBS-MEF and MCF7 cells by
trypsinization for 5 min (we recommend using 3 mL trypsinEDTA added per plate) at 37
C.
3. Neutralize trypsin-EDTA by adding 3–5 mL of complete
DMEM medium.
4. Pass the cell suspension through a 40 μm cell strainer.
5. Count cells by a hemocytometer or cell counter and mix
3 Â 10
6 cells of each cell type in a pre-chilled 15 mL tube (see
Note 4).
6. (Optional, but highly recommended) At this point, in order to
check for the efficiency of sorting, an aliquot of approximately
5–10 Â 10
4 of the “mix” cell populations can be saved for
analysis by flow cytometry.
7. Spin down the cells in a centrifuge at 350 Â g for 10 min at 4
C
and aspirate the medium carefully.
8. Resuspend the cells in 400 μL of cold DMEM and proceed to
magnetic bead-mediated cell sorting.
3.1.3 Preparation
of “Co-culture” Samples
1. Harvest one cell type, which can be designated as the “acceptor” cells, from a standing culture (e.g., here we chose MCF7
cells, see Note 5). Following trypsinization, neutralize by adding complete DMEM, and count the cells using a hemocytometer or automatic cell counter (see Note 6).
2. Add 3 Â 10
6 MCF7 cells to 20 mL complete medium in a
fibronectin-coated dish and incubate for 3–4 h.
3. After 3–4 h, add 3 Â 10
6 cells of the “donor” line (e.g., here we
chose the MBS-MEFs) to the same dish and co-culture for the
required duration of time. For example, we found that 12 h of
co-culture leads to the transfer of ~2% of β-actin-MBS mRNA
from MBS-MEFs to MCF7 cells (see Note 7).
Use of RNA Tagging as a Control for RNA Transferome Analysis
201
of Culture Medium
and Fibronectin-Coated
Plates
1. Prepare complete culture medium to reach a final concentration of 10% FBS, 1% sodium pyruvate solution, and 1%
penicillin-streptomycin solution in DMEM.
2. Coat the 150 mm dishes with fibronectin: Rinse the 150 mm
dishes with 10 mL PBS. Add 10 mL of the fibronectin working
solution to each dish and incubate at 37
C for 20 min. After
incubation, rinse the dish with 10 mL PBS.
3.1.2 Preparation
of “Mix” Control Samples
As a control for the transfer of RNA between cells in co-culture, we
strongly recommend having a negative control of the two cell types
mixed together (“mix”) and immediately sorted (see Subheading
3.2). Proceed as follows:
1. Culture the different cell types in fibronectin-coated dishes,
prepared as described in Subheading 3.1.1.
2. Harvest the separately cultured MBS-MEF and MCF7 cells by
trypsinization for 5 min (we recommend using 3 mL trypsinEDTA added per plate) at 37
C.
3. Neutralize trypsin-EDTA by adding 3–5 mL of complete
DMEM medium.
4. Pass the cell suspension through a 40 μm cell strainer.
5. Count cells by a hemocytometer or cell counter and mix
3 Â 10
6 cells of each cell type in a pre-chilled 15 mL tube (see
Note 4).
6. (Optional, but highly recommended) At this point, in order to
check for the efficiency of sorting, an aliquot of approximately
5–10 Â 10
4 of the “mix” cell populations can be saved for
analysis by flow cytometry.
7. Spin down the cells in a centrifuge at 350 Â g for 10 min at 4
C
and aspirate the medium carefully.
8. Resuspend the cells in 400 μL of cold DMEM and proceed to
magnetic bead-mediated cell sorting.
3.1.3 Preparation
of “Co-culture” Samples
1. Harvest one cell type, which can be designated as the “acceptor” cells, from a standing culture (e.g., here we chose MCF7
cells, see Note 5). Following trypsinization, neutralize by adding complete DMEM, and count the cells using a hemocytometer or automatic cell counter (see Note 6).
2. Add 3 Â 10
6 MCF7 cells to 20 mL complete medium in a
fibronectin-coated dish and incubate for 3–4 h.
3. After 3–4 h, add 3 Â 10
6 cells of the “donor” line (e.g., here we
chose the MBS-MEFs) to the same dish and co-culture for the
required duration of time. For example, we found that 12 h of
co-culture leads to the transfer of ~2% of β-actin-MBS mRNA
from MBS-MEFs to MCF7 cells (see Note 7).
Use of RNA Tagging as a Control for RNA Transferome Analysis
201