corresponding to the “mix” and “co-culture” samples can be
saved for analysis by flow cytometry.
11. Place the tubes on ice and spin down the cells at 350 Â g for
10 min at 4
C.
12. Aspirate the supernatant and flash-freeze until RNA extraction
or proceed to RNA extraction immediately.
13. Purify total RNA using a kit of choice according to the manufacturer’s instructions (see Note 9). Quantify total RNA using a
Nano Drop microvolume spectrophotometer. Treatment with
DNase is NOT recommended if intending to check the sorting
efficiency later (see Subheading 3.3.2).
3.3 Quality Control
for Cell Sorting
We recommend two methods to determine the efficiency of sorting—by flow cytometry and by DNA-PCR.
3.3.1 Quality Control
for Cell Sorting by Flow
Cytometry
As mentioned in the previous section, aliquots of samples—“mix”
and “co-culture”—obtained both before and after sorting can be
saved for analysis by flow cytometry. It is helpful if the cells used have
a genetic fluorescent marker (e.g., GFP, RFP). If such cell line is not
available, the cells can be counterstained using an antibody against a
specific surface marker suitable for flow cytometry. If both cells
express fluorescently labeled proteins (i.e., in two different excitation
and emission spectra), they can be directly analyzed by a flow cytometer. If even one type of the cells requires labeling with an
antibody, the following steps can be followed as described below:
1. Prepare cell suspensions having between 5 and 10 Â 10
5 cells in
50–100 μL of complete culture (DMEM) medium or sorting
buffer.
2. Add the first antibody (per the recommended dilution) and
incubate on ice for 30 min (if the first antibody is fluorescently
labeled, incubation should be performed in the dark).
3. Spin down the cells at 350 Â g for 5 min at 4
C. Wash twice by
resuspending the cells in 500 μL of cold medium and spinning
down at 350 Â g for 5 min at 4
C after each wash.
4. Add the second antibody (per the recommended dilution) and
incubate on ice for 30 min in the dark.
5. Spin down the cells at 350 Â g for 5 min at 4
C. Wash once by
resuspending in 500 μL of cold medium and spinning down at
350 Â g for 5 min at 4
C. Resuspend the cells in 500 μl of cold
medium.
6. Incubate with 0.25 μg PI or 7-AAD per 10
6 cells for 10 min at
room temperature.
7. Transfer to FACS tubes and analyze on a flow cytometer. See
Fig. 3a for representative results.
Use of RNA Tagging as a Control for RNA Transferome Analysis
203
saved for analysis by flow cytometry.
11. Place the tubes on ice and spin down the cells at 350 Â g for
10 min at 4
C.
12. Aspirate the supernatant and flash-freeze until RNA extraction
or proceed to RNA extraction immediately.
13. Purify total RNA using a kit of choice according to the manufacturer’s instructions (see Note 9). Quantify total RNA using a
Nano Drop microvolume spectrophotometer. Treatment with
DNase is NOT recommended if intending to check the sorting
efficiency later (see Subheading 3.3.2).
3.3 Quality Control
for Cell Sorting
We recommend two methods to determine the efficiency of sorting—by flow cytometry and by DNA-PCR.
3.3.1 Quality Control
for Cell Sorting by Flow
Cytometry
As mentioned in the previous section, aliquots of samples—“mix”
and “co-culture”—obtained both before and after sorting can be
saved for analysis by flow cytometry. It is helpful if the cells used have
a genetic fluorescent marker (e.g., GFP, RFP). If such cell line is not
available, the cells can be counterstained using an antibody against a
specific surface marker suitable for flow cytometry. If both cells
express fluorescently labeled proteins (i.e., in two different excitation
and emission spectra), they can be directly analyzed by a flow cytometer. If even one type of the cells requires labeling with an
antibody, the following steps can be followed as described below:
1. Prepare cell suspensions having between 5 and 10 Â 10
5 cells in
50–100 μL of complete culture (DMEM) medium or sorting
buffer.
2. Add the first antibody (per the recommended dilution) and
incubate on ice for 30 min (if the first antibody is fluorescently
labeled, incubation should be performed in the dark).
3. Spin down the cells at 350 Â g for 5 min at 4
C. Wash twice by
resuspending the cells in 500 μL of cold medium and spinning
down at 350 Â g for 5 min at 4
C after each wash.
4. Add the second antibody (per the recommended dilution) and
incubate on ice for 30 min in the dark.
5. Spin down the cells at 350 Â g for 5 min at 4
C. Wash once by
resuspending in 500 μL of cold medium and spinning down at
350 Â g for 5 min at 4
C. Resuspend the cells in 500 μl of cold
medium.
6. Incubate with 0.25 μg PI or 7-AAD per 10
6 cells for 10 min at
room temperature.
7. Transfer to FACS tubes and analyze on a flow cytometer. See
Fig. 3a for representative results.
Use of RNA Tagging as a Control for RNA Transferome Analysis
203