2. Thaw frozen cells at room temperature before proceeding with
RNA extraction. We follow the protocol as indicated by the
supplier with the following specificities: (a) we do not add
β-mercaptoethanol nor DTT into the RLT buffer, (b) we skip
the DNase digestion optional step, and (c) we do all centrifugation steps at 9000 Â g.
3. Elute in 30 μL RNase-free water.
4. You can directly proceed with quantification (see Note 43) and
reverse transcription (recommended) or keep at À20
C for
short storage (up to 1 year). For long storage (more than
1 year), keep at À80
C. Always thaw on ice and avoid
freeze/thaw cycles of the RNA.
Perform reverse transcription (see steps 5–11) working on
ice, in 250 μL PCR tubes (see Note 44).
5. Distribute 1500 ng RNA per tube in up to 13.75 μL nucleasefree water from the kit.
6. Add, per tube, 1.25 μL of mix 1, previously prepared and kept
on ice. Mix gently and centrifuge briefly.
7. Denature components 5 min at 65
C in PCR machine. Meanwhile, prepare mix 2 and keep on ice.
8. Chill PCR tube on ice and add 5 μL per tube of mix 2. Mix
gently and centrifuge briefly.
9. Incubate 10 min at 25
C followed by 30 min at 65
C and
5 min at 85
C in PCR machine.
10. Chill on ice and directly proceed to qPCR or store at À20
C
for up to 1 week, or at À80
C for longer storage. Avoid
freeze/thaw cycles of the RNA.
11. Proceed with cDNA/RNA hybrid quantification by measuring
A 260 on a spectrophotometer, considering that A 260 ¼ 1 corresponds to a cDNA/RNA hybrid concentration of 50 ng/μL
(see Note 45).
Perform qPCR following steps 12–17 (see Note 46). All
volumes indicated are for 384-well qPCR plates. Controls
ahead of the qPCR are required (see Note 47).
Primers should be designed on exon-exon boundaries in
order to avoid genomic DNA amplification (see Table 2).
12. Prepare all dilutions and keep on ice (see Note 48).
13. Use an electronic device and distribute cDNA first, then the
primers in SYBR green mix. This will avoid crosscontamination since the SYBR green sticks to the well. From
time to time, tap the plate onto the bench to enable droplets to
slide to the bottom of the well. The final volume in the wells is
6 μL.
EMT Studied by Molecular Biology and DNA-FISH in Human Cells
367
RNA extraction. We follow the protocol as indicated by the
supplier with the following specificities: (a) we do not add
β-mercaptoethanol nor DTT into the RLT buffer, (b) we skip
the DNase digestion optional step, and (c) we do all centrifugation steps at 9000 Â g.
3. Elute in 30 μL RNase-free water.
4. You can directly proceed with quantification (see Note 43) and
reverse transcription (recommended) or keep at À20
C for
short storage (up to 1 year). For long storage (more than
1 year), keep at À80
C. Always thaw on ice and avoid
freeze/thaw cycles of the RNA.
Perform reverse transcription (see steps 5–11) working on
ice, in 250 μL PCR tubes (see Note 44).
5. Distribute 1500 ng RNA per tube in up to 13.75 μL nucleasefree water from the kit.
6. Add, per tube, 1.25 μL of mix 1, previously prepared and kept
on ice. Mix gently and centrifuge briefly.
7. Denature components 5 min at 65
C in PCR machine. Meanwhile, prepare mix 2 and keep on ice.
8. Chill PCR tube on ice and add 5 μL per tube of mix 2. Mix
gently and centrifuge briefly.
9. Incubate 10 min at 25
C followed by 30 min at 65
C and
5 min at 85
C in PCR machine.
10. Chill on ice and directly proceed to qPCR or store at À20
C
for up to 1 week, or at À80
C for longer storage. Avoid
freeze/thaw cycles of the RNA.
11. Proceed with cDNA/RNA hybrid quantification by measuring
A 260 on a spectrophotometer, considering that A 260 ¼ 1 corresponds to a cDNA/RNA hybrid concentration of 50 ng/μL
(see Note 45).
Perform qPCR following steps 12–17 (see Note 46). All
volumes indicated are for 384-well qPCR plates. Controls
ahead of the qPCR are required (see Note 47).
Primers should be designed on exon-exon boundaries in
order to avoid genomic DNA amplification (see Table 2).
12. Prepare all dilutions and keep on ice (see Note 48).
13. Use an electronic device and distribute cDNA first, then the
primers in SYBR green mix. This will avoid crosscontamination since the SYBR green sticks to the well. From
time to time, tap the plate onto the bench to enable droplets to
slide to the bottom of the well. The final volume in the wells is
6 μL.
EMT Studied by Molecular Biology and DNA-FISH in Human Cells
367