3.6 Real-Time PCR
Analysis
1. At the end of each EMT or MET phases, pellet 500,000 cells,
snap freeze and store at À80
C.
2. Extract total RNAs using PureLink RNA kit according to the
manufacturer’s instructions.
3. Perform cDNA synthesis using High-Capacity cDNA Reverse
Transcription kit (Applied Biosystems) with Oligo(dT) primers
(ThermoFisher). Use 500 ng of total RNA for transcription.
Perform real-time qPCR using FastStart Universal SYBR
Green Master mix (Roche) and primers specifically designed
to amplify SNAIL1, SNAIL2 and ZEB1 (see Note 7). For each
sample, perform qPCRs with experimental replicates, and melting curve analysis is performed for each run. Final results
represent relative gene expression values calculated with the
ΔΔCt method using the geometric mean of three reference
genes for normalization [13] and control MCF10A as the
reference sample (Fig. 3b).
4 Notes
1. Cell types: these experiments were performed with the
MCF10A cell line [11]. The choice of the cell line depends
on the biological issue to be addressed, on the ability of the
cells to respond to EMT induction and then to return to the
epithelial state and on the cell toxicity of EMT inducing
molecules.
2. Culture media: in order to avoid bias in the reproducibility of
the cycles, it is preferable to prepare fresh EMT-IM and control
medium for each cycle.
3. Cytokine choice: the choice of the cytokines to induce EMT
depends on the cells used. The combination of TGF-β,
TNF-α and IL-6, as three cytokines known to induce EMT,
was a well permissive condition on MCF10A, as well as TGF- β
alone.
4. Estimate of the number of dishes: usually, for each experiment,
each cycle and each experimental condition, we prepare five
60 mm culture dishes: one for performing flow cytometry, one
for keeping cell pellets, two (with coverslips) for immunostaining and two to be used to seed cells for the next cycle.
5. Cell density: seeding cell density is a crucial point that has to be
optimized. A high cell density can inhibit or slow down EMT
induction whereas a low cell density can lead to not having
enough cells at the end of the cycle to reseed and carry out the
various planned analyses. Indeed, EMT induction significantly
decreases cell proliferation [14].
Inducing Sequential Cycles of EMT and MET in Epithelial Cells
349
Analysis
1. At the end of each EMT or MET phases, pellet 500,000 cells,
snap freeze and store at À80
C.
2. Extract total RNAs using PureLink RNA kit according to the
manufacturer’s instructions.
3. Perform cDNA synthesis using High-Capacity cDNA Reverse
Transcription kit (Applied Biosystems) with Oligo(dT) primers
(ThermoFisher). Use 500 ng of total RNA for transcription.
Perform real-time qPCR using FastStart Universal SYBR
Green Master mix (Roche) and primers specifically designed
to amplify SNAIL1, SNAIL2 and ZEB1 (see Note 7). For each
sample, perform qPCRs with experimental replicates, and melting curve analysis is performed for each run. Final results
represent relative gene expression values calculated with the
ΔΔCt method using the geometric mean of three reference
genes for normalization [13] and control MCF10A as the
reference sample (Fig. 3b).
4 Notes
1. Cell types: these experiments were performed with the
MCF10A cell line [11]. The choice of the cell line depends
on the biological issue to be addressed, on the ability of the
cells to respond to EMT induction and then to return to the
epithelial state and on the cell toxicity of EMT inducing
molecules.
2. Culture media: in order to avoid bias in the reproducibility of
the cycles, it is preferable to prepare fresh EMT-IM and control
medium for each cycle.
3. Cytokine choice: the choice of the cytokines to induce EMT
depends on the cells used. The combination of TGF-β,
TNF-α and IL-6, as three cytokines known to induce EMT,
was a well permissive condition on MCF10A, as well as TGF- β
alone.
4. Estimate of the number of dishes: usually, for each experiment,
each cycle and each experimental condition, we prepare five
60 mm culture dishes: one for performing flow cytometry, one
for keeping cell pellets, two (with coverslips) for immunostaining and two to be used to seed cells for the next cycle.
5. Cell density: seeding cell density is a crucial point that has to be
optimized. A high cell density can inhibit or slow down EMT
induction whereas a low cell density can lead to not having
enough cells at the end of the cycle to reseed and carry out the
various planned analyses. Indeed, EMT induction significantly
decreases cell proliferation [14].
Inducing Sequential Cycles of EMT and MET in Epithelial Cells
349