Pulse-Chase mRNA decay assay in HeLa Tet-off cells:
This protocol is for four time-points in 12-well plates. Scale up or down as needed.
Day 1: Plate cells:
Split one or more 10-cm plate(s) with 80-100% confluent HeLa Tet-off cells to a final volume of 10-11 ml. Make sure to break up cell clusters to ensure single cells.
Dilute freshly split cell suspension 1:12 or 1:15 (depending on % confluency) in DMEM/10% FBS in a 50 ml conical in a total volume equal to or more than the number of wells to be plated (for e.g. make about 50 ml for 48 wells -12 experiments of 4 time-points).
Plate 1 ml per well in 12-well plates.
Incubate over night.
Day 2: Transfections
Using Mirus TransIT HeLaMonster (check manufacturers protocols if using other reagents)
Cells should be 40-60% confluent - not more.
For each of four wells:
1) Add a total of 4.5 µg plasmid to an eppendorf tube:
For B-globin reporters use:
45 ng pcBwtGAP3 UAC (internal control mRNA)
0.9 µg pPC or pcTET2 B-globin reporter
3.6 µg of other plasmid(s) - such as protein expressing or empty vectors
Optional: If using same reporters in all experiments, make a larger mix of internal control and B-globin reporter. For e.g., for 12 experiments, mix
45 ng x 12.5 of pcBwtGAP3 UAC (internal control mRNA)
0.9 µg x 12.5 of pPC or pcTET2 B-globin reporter
Then, divide the mix into 12.5 equal parts and pipet into separate eppendorfs.
2) Mix 225 µl O-MEM with 13.5 µl TransIT HeLa reagent
Add 0.225 µl of 1 mg/ml Tetracycline (in EtOH).
(a final concentration of 50 ng/ml Tet ensures no expression from the TET-driven promoter)
Incubate 5-20 mins at RT
3) Mix 5 µl HeLaMonster reagent with 45 µl ddw. Store on ice.
(Important: Thaw HeLaMonster reagent just before use, keep on ice and return to freezer immediately).
Optional: Bigger mixes can be made by multiplying O-MEM, TransIT reagent and Tetracycline volumes (Step 2) or HeLaMonster and ddw volumes (Step 3) by the number of experiments (+0.5).
4) Add 225 µl O-MEM/TransIT HeLa mix to the plasmid mix.
Incubate 5-20 mins at RT
5) To each 2.0-cm well of HeLa Tet-off cells:
Add 50 µl of O-MEM/TransIT/Plasmid mix.
6) To each 2.0-cm well of HeLa Tet-off cells:
Add 10 µl of diluted HeLaMonster reagent.
Place plates in incubator.
Day 4: Pulse-Chase assay.
1) Pulse transcription (Early morning):
Start transcription from Tet-promoter:
Wash wells carefully (!) with 1 ml PBS.
Add 1 ml DMEM/10% FBS.
Incubate Å6 hours
Stop transcription (the complete stop will take Å20 mins):
To each well, add 10 µl of O-MEM/100 µg/ml Tetracycline
Place back in incubator.
3) Time points:
Take the first time point 20-30 mins after addition of tetracycline (t=0).
For each time point:
Wash cells with 1 ml PBS.
Add 0.5 ml Trizol. Pipet up and down until non-viscous.
Transfer to pre-labeled eppendorf tube.
Store in -20 C.
Day 5: RNA preps:
To each tube with 0.5 ml Trizol:
Add 100 µl Chloroform.
Mix by shaking for 30 sec or more.
Leave at RT, 10 min.
Spin 12,000 rpm, Å10 min.
Transfer 250 µl supernatant to new tube. Avoid interphase!!
Add 0.7 volumes of isopropanol (175 µl). Mix and leave at RT, 10 min.
Spin 12,000 g (not faster!), 4C, 10 min.
Remove supernatant carefully.
Wash carefully with 0.5 ml of 70% EtOH. Spin at 7,500 g for 5 min, 4C.
Dissolve in 10 µl formamide load buffer.
Run 5 µl on a Northern Gel.